Methods and compositions relating to anti-chi3l1 antibody reagents

ABSTRACT

It is demonstrated herein that inhibitors of immune checkpoints and CHI3L1 are synergistic. Accordingly, described herein are methods and compositions relating to combinatorial therapies for cancer, e.g., comprising an inhibitor of CHI3L1; and an inhibitor of an immune checkpoint protein. In some embodiments, the CHI3L1 inhibitor can be an antibody or antibody reagent as described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 365(c) and 35 U.S.C. §120 of International Application No. PCT/US18/047633 filed Aug. 23,2018, which designates the U.S. and which claims benefit under 35 U.S.C.§ 119(e) of U.S. Provisional Application Nos. 62/650,374 filed Mar. 30,2018 and 62/549,043 file Aug. 23, 2017, the contents of which areincorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos. UH2 HL123876 and UO1 HL108638 awarded by the National Institutes of Health.The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 23, 2018, isnamed 058040-091590WOPT_SL.txt and is 115,924 bytes in size.

TECHNICAL FIELD

The technology described herein relates to treatments for cancer.

BACKGROUND

Lung cancer afflicts over 1.8 million people annually, with 1.6 yearlydeaths. It is the leading cause of cancer-related death in men and has alow long-term survival rate. Developing improved treatments for thisdisease is a major public health concern.

SUMMARY

It is demonstrated herein that inhibiting a) CHI3L1 and/or CHI3L1signaling and b) at least one immune checkpoint protein providessynergistic effects in the treatment of cancer, e.g., lung cancer.Accordingly, provided herein are methods and compositions related to thetreatment of cancer.

In one aspect of any of the embodiments, described herein is a method oftreating cancer in a subject in need thereof, the method comprisingadministering: an inhibitor of CHI3L1; and an inhibitor of an immunecheckpoint protein.

In one aspect of any of the embodiments, described herein is atherapeutically effective amount of an inhibitor of CHI3L1 and aninhibitor of an immune checkpoint protein for use in the treatment ofcancer. In some embodiments of any of the aspects, the inhibitor ofCHI3L1 and the inhibitor of an immune checkpoint protein are present inthe same composition. In some embodiments of any of the aspects, theinhibitor of CHI3L1 and the inhibitor of an immune checkpoint proteinare present in separate compositions.

In one aspect of any of the embodiments, described herein is apharmaceutical composition comprising an inhibitor of CHI3L1; and aninhibitor of an immune checkpoint protein. In one aspect of any of theembodiments, described herein is a kit comprising a pharmaceuticalcomposition comprising an inhibitor of CHI3L1; and a pharmaceuticalcomposition comprising an inhibitor of an immune checkpoint protein.

In some embodiments of any of the aspects, the inhibitor of CHI3L1 is anantibody, antibody reagent, antigen-binding fragment thereof, or CARthat specifically binds to CHI3L1.

In some embodiments of any of the aspects, the antibody, antibodyreagent, antigen-binding fragment thereof, or CAR comprises at least oneheavy or light chain complementarity determining region (CDR) selectedfrom the group consisting of:

-   -   (a) a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 4;    -   (b) a light chain CDR2 having the amino acid sequence of SEQ TD        NO: 5;    -   (c) a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 6;    -   (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 1;    -   (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 2; and    -   (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 3; or

a conservative substitution variant of one or more of (a)-(f).

In some embodiments of any of the aspects, the antibody, antibodyreagent, antigen-binding portion thereof, or CAR of comprises heavychain CDRs having the amino acid sequences of SEQ ID NOs: 1-3 or aconservative substitution variant of such amino acid sequence. In someembodiments of any of the aspects, the antibody, antibody reagent,antigen-binding portion thereof or CAR comprises light chain CDRs havingthe amino acid sequences of SEQ ID NOs: 4-6 or a conservativesubstitution variant of such amino acid sequence. In some embodiments ofany of the aspects, the antibody, antibody reagent, antigen-bindingportion thereof or CAR comprises light chain CDRs having the amino acidsequences of SEQ ID NOs: 4-6 and heavy chain CDRs having the amino acidsequences of SEQ ID NOs: 1-3 or a conservative substitution variant ofsuch amino acid sequence.

In some embodiments of any of the aspects, the inhibitor of CHI3L1 isantibody, antibody reagent, antigen-binding portion thereof or CAR thatcompetes for binding to CHI3L1 with an antibody comprising light chainCDRs having the amino acid sequences of SEQ ID NOs: 4-6 and heavy chainCDRs having the amino acid sequences of SEQ ID NOs: 1-3.

In some embodiments of any of the aspects, the inhibitor of CHI3L1 bindsa CHI3L1 polypeptide at an epitope selected from SEQ ID NOs: 13-24.

In some embodiments of any of the aspects, the inhibitor of CHI3L1 is aninhibitor of a CHI3L1 receptor. In some embodiments of any of theaspects, the CHI3L1 receptor is IL13Rα2, TMEM219, or CRTH2.

In some embodiments of any of the aspects, the inhibitor is aninhibitory antibody or nucleic acid molecule.

In some embodiments of any of the aspects, the cancer is a primarycancer or a metastatic cancer. In some embodiments of any of theaspects, the cancer is malignant cancer. In some embodiments of any ofthe aspects, the cancer is selected from the group consisting of:prostate cancer, colon cancer, rectal cancer, ovarian cancer, kidneycancer, breast cancer, glioblastoma, melanoma, malignant melanoma, andlung cancer.

In some embodiments of any of the aspects, the inhibitor of an immunecheckpoint protein is an antibody, antibody reagent, antigen-bindingfragment thereof, or CAR that specifically binds to at least one immunecheckpoint protein. In some embodiments of any of the aspects, theinhibitor of an immune checkpoint protein is a natural ligand thereof.In some embodiments of any of the aspects, the natural ligand comprisesPD-L1 or B7. In some embodiments of any of the aspects, the immunecheckpoint protein is selected from the group consisting of: PD-1;PD-L1; PD-L2; TIM-3; CTLA4; TIGIT; DD1-α; A2AR; B7-H3; B7-H4; BTLA; IDO;TDO; KIR; and LAGS. In some embodiments of any of the aspects, theinhibitor of an immune checkpoint protein is selected from the groupconsisting of: MGA271; ipilimumab; pembrolizumab; nivolumab;atezolizumab; galiximab; IMP321; BMS-986016; SMB-663513; PF-05082566;IPH2101; KW-0761; CDX-1127; MEDI-6769; CP-870,893; tremelimumab;pidilizumab; MPDL3280A; MED14736; MSB0010718C; AUNP12; avelumab; anddurvalumab.

In some embodiments of any of the aspects, the inhibitor of CHI3L1 andthe inhibitor of an immune checkpoint protein are present in the samebivalent antibody reagent. In some embodiments of any of the aspects,the immune checkpoint protein is PD-1 or CTLA4.

In some embodiments of any of the aspects, the subject is a subjectdetermined to have an elevated level of CHI3L1. In some embodiments ofany of the aspects, the CHI3L1 is circulating CHI3L1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict the characterization of the FRG monoclonal antibody(mAb). FIG. 1A demonstrates mAb analysis under non-denaturing (Non-Den)and denaturing (DEN) conditions with Coomassie staining, Western blotand Isotyping. FIG. 1B depicts FRG detection of Chi3l1 in non-denaturingand denaturing conditions. FIG. 1C depicts Sensitivity and specifcity ofFRG against recombinant (r) human and mouse Chi3l1 detected by Westernblot. FIG. 1D depicts FRG affinity and dose response curve evaluated byELISA.

FIGS. 2A-2B demonstrate the neutralizing effects of FRG onChi3l1-stimulated signaling. FIG. 2A depicts effects on ERK and AkKTphosphorylation in peritoneal macrophages*. FIG. 2B depicts the effectsof different doses of FRG on ERK and AKT phosphorylation in onperitoneal macrophages *. * Thp1 cells, U937 cells, and AMJ2-C11 (mousealveolar macrophages cell line) showed similar pattern of inhibition anddose responses on Chi3l1-stimulated Erk and Akt activation.

FIG. 3 depicts graphs of the effects of FRG on aeroallergen-stimulatedtype 2 lung inflammation.

FIG. 4 depicts the effects of FRG on melanoma metastasis. Mice receivedthe noted doses of B16-F10 cells by tail vein injection and were treatedwith control IGg2b antibody or FRG (200 μg/per mouse) every other day.The mice were sacrificed after 14 days and pulmonary metastasis (sarkdots) were evaluated.

FIGS. 5A-5B demonstrate the effects of FRG on primary lung cancer. FIG.5A depicts the tunor accumulation in representative lungs from mice withboth activating KRAS^(G12D) mutations and null p53 mutations which weretreated with control (Ctrl) IgG or FRG mAb. FIG. 5B depicts arepresentative histology of the lungs illustrated in FIG. 5A.

FIG. 6 depicts the location of selected epitopes including FRG in humanChi3l1.

FIG. 7 depicts the light chain CDR sequences of the FRG antibodydescribed herein (SEQ ID NOS 55 and 56, respectively, in order ofappearance).

FIG. 8 depicts the heavy chain CDR sequences of the FRG antibodydescribed herein (SEQ ID NOS 57 and 58, respectively, in order ofappearance).

FIG. 9 depicts B16F10 melanoma metastasis in mice treated with the FRGantibody described herein in combination with Anti-PD-1. Images shownwere obtained on Day 14. In these experiments malignant melanocytes wereadministered by tail vein to wild type (WT) mice that were treated everyother day with the noted dosages of FRG and or anti-PD-1, alone and incombination, via an intraperitoneal route. Fourteen days later the lungswere removed and the levels of metastasis (black dots on pleuralsurface) were assessed. The beneficial interactions of FRG and anti-PD-1in combination can be seen.

FIG. 10 depicts a graph of the quantitation of the data shown in FIG. 9.

DETAILED DESCRIPTION

Described herein are combinatorial treatments for cancer, e.g., usingthe synergistic effect of targeting both CHI3L1 and immune checkpoints.Such synergistic effects are demonstrated herein. Additionally, CHI3L1is regulated by the interaction of Sema7A with β1-integrin and PlexinC1.Specifically, Sema7A bound to β1-integrin stimulates CHI3L1 expressionand activity, while Sema7A bound to PlexinC1 inhibits the expression andactivity of CHI3L1. Thus, CHI3L1 can be inhibited by directly targetingCHI3L1, indirectly targeting CHI3L1 (e.g., by targeting its receptors),or by inhibiting semaphorin 7a (Sema7A), inhibiting β1-integrin, oragonizing PlexinC1.

Accordingly, in one aspect of any of the embodiments, provided herein isa method of treating cancer in a subject in need thereof, the methodcomprising administering a) an inhibitor of an immune checkpoint proteinand b) at least one of: an inhibitor of CHI3L1, an inhibitor of Sema7A,an inhibitor of β1-integrin, and/or an agonist of PlexinC1. In someembodiments of any of the aspects, the method comprises administering a)an inhibitor of an immune checkpoint protein and b) two or more of: aninhibitor of CHI3L1, an inhibitor of Sema7A, an inhibitor ofβ1-integrin, and/or an agonist of PlexinC1 in combination with aninhibitor of an immune checkpoint protein. In some embodiments of any ofthe aspects, the method comprises administering, a) an inhibitor of animmune checkpoint protein and b) three or more of an inhibitor ofCHI3L1, an inhibitor of Sema7A, an inhibitor of β1-integrin, and/or anagonist of PlexinC1. In some embodiments of any of the aspects, themethod comprises administering, in combination with an inhibitor of animmune checkpoint protein, all of an inhibitor of CHI3L1, an inhibitorof Sema7A, an inhibitor of β1-integrin, and/or an agonist of PlexinC1.Table 4 depicts exemplary but non-limiting examples of pairwisecombinations of the agents which can be administered in the foregoingmethods, e.g., in combination with an inhibitor of an immune checkpointprotein.

TABLE 4 INHIBITOR INHIBITOR AGONIST INHIBITOR OF OF B1- OF OF CHI3L1SEMA7A INTEGRIN PLEXINC1 INHIBITOR X X X OF CHI3L1 INHIBITOR X X X OFSEMA7A INHIBITOR X X X OF B1- INTEGRIN AGONIST OF X X X PLEXINC1

In some embodiments of any of the aspects two or more individualreagents of each of the categories of an inhibitor of an immunecheckpoint protein, an inhibitor of CHI3L1, an inhibitor of Sema7A, aninhibitor of β1-integrin, and/or an agonist of PlexinC1 can beadministered, e.g., two different inhibitors of an immune checkpointprotein or two different Sema7A inhibitors.

In one aspect of any of the embodiments, provided herein is a method oftreating cancer in a subject in need thereof, the method comprisingadministering an inhibitor of CHI3L1; and an inhibitor of an immunecheckpoint protein.

As used herein, “inhibitor” refers to an agent which can decrease theexpression and/or activity of a target, e.g. by at least 10% or more,e.g. by 10% or more, 50% or more, 70% or more, 80% or more, 90% or more,95% or more, or 98% or more. The efficacy of an inhibitor of one or moretargets, e.g. its ability to decrease the level and/or activity of thetarget can be determined, e.g. by measuring the level of an expressionproduct of the target and/or the activity of the target. Methods formeasuring the level of a given mRNA and/or polypeptide are known to oneof skill in the art, e.g. RT-PCR with primers can be used to determinethe level of RNA and Western blotting with an antibody can be used todetermine the level of a polypeptide. The activity of, e.g. CHI3L1, canbe determined using methods known in the art. In some embodiments of anyof the aspects, the inhibitor can be an inhibitory nucleic acid; anaptamer; an antibody reagent; an antibody; or a small molecule. Aninhibitor of a target described herein can inhibit the activity,expression, or accumulation of the target polypeptide.

Inhibitors of, e.g., CHI3L1, can include inhibitors that act directly onthe target itself (e.g., that bind to the CHI3L1 protein or transcript,e.g., direct inhibitors) or inhibitors that act indirectly on thetarget, e.g., directly on one or more receptors for CHI3L1. As usedherein, “CHI3L1,” “chintinase-3-like-1 protein,” or “YKL-40” refers to a˜40 kDa glycoprotein secreted by at least macrophages, chondrocytes,neutrophils, synovial cells, and some cancer cells. CHI3L1 does not havechitinase activity, is a Th2 promoting cytokine, has been linked to theAKT anti-apoptotic signaling pathway and induces the migration ofastrocytes. The sequences of CHI3L1 expression products are known for anumber of species, e.g., human CHI3L1 (NCBI Gene ID No: 1116) mRNA (SEQID NO: 59; NCBI Ref Seq: NM_001276.1 and SEQ ID NO: 26; NCBI Ref Seq:NM_001276.2) and polypeptide (SEQ ID NO: 27; NCBI Ref Seq: NP_001267.1and SEQ ID NO: 28; NCBI Ref Seq: NP_001267.2). Chitinase 3-like-1 isreferred to in the art as Chil1.

In some embodiments of any of the aspects the inhibitor of CHI3L1 ispoly (I:C), which is known in the art and described in more detail at,e.g., Ma et al. Scientific Reports 2016 6:26299; which is incorporatedby reference herein in its entirety.

In some embodiments of any of the aspects, an inhibitor of a specifiedtarget is an antibody, antibody reagent, antigen-binding fragmentthereof, or CAR that specifically binds to the target. In someembodiments of any of the aspects, the inhibitor of CHI3L1 is anantibody, antibody reagent, antigen-binding fragment thereof, or CARthat specifically binds to CHI3L1.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The term also refers to antibodies comprised of twoimmunoglobulin heavy chains and two immunoglobulin light chains as wellas a variety of forms including full length antibodies andantigen-binding portions thereof; including, for example, animmunoglobulin molecule, a monoclonal antibody, a chimeric antibody, aCDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, aFv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), adiabody, a multispecific antibody, a dual specific antibody, ananti-idiotypic antibody, a bispecific antibody, a functionally activeepitope-binding portion thereof, and/or bifunctional hybrid antibodies.

Each heavy chain is composed of a variable region of said heavy chain(abbreviated here as HCVR or VH) and a constant region of said heavychain. The heavy chain constant region consists of three domains CH1,CH2 and CH3. Each light chain is composed of a variable region of saidlight chain (abbreviated here as LCVR or VL) and a constant region ofsaid light chain. The light chain constant region consists of a CLdomain. The VH and VL regions may be further divided into hypervariableregions referred to as complementarity-determining regions (CDRs) andinterspersed with conserved regions referred to as framework regions(FR). Each VH and VL region thus consists of three CDRs and four FRswhich are arranged from the N terminus to the C terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure iswell known to those skilled in the art.

As used herein, the term “CDR” refers to the complementarity determiningregions within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain and of the light chain, whichare designated CDR1, CDR2 and CDR3, for each of the variable regions.The exact boundaries of these CDRs have been defined differentlyaccording to different systems. The system described by Kabat (Kabat etal., Sequences of Proteins of Immunological Interest (NationalInstitutes of Health, Bethesda, Md. (1987) and (1991)) not only providesan unambiguous residue numbering system applicable to any variableregion of an antibody, but also provides precise residue boundariesdefining the three CDRs. These CDRs may be referred to as Kabat CDRs.Other boundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J MolBiol 262(5):732-45 (1996)) and Chothia (J. Mol. Biol. 196:901-917 (1987)and Nature 342:877-883 (1989)). Still other CDR boundary definitions maynot strictly follow one of the above systems, but will nonethelessoverlap with the Kabat CDRs, although they may be shortened orlengthened in light of prediction or experimental findings thatparticular residues or groups of residues or even entire CDRs do notsignificantly impact antigen binding. The methods used herein mayutilize CDRs defined according to any of these systems, althoughpreferred embodiments use Kabat defined CDRs. The CDR's identifiedherein, e.g., SEQ ID NOs 1-6 are identified by the Kabat system (see,e.g. FIGS. 7 and 8).

The term “antigen-binding portion” of an antibody refers to one or moreportions of an antibody as described herein, said portions) still havingthe binding affinities as defined above herein. Portions of a completeantibody have been shown to be able to carry out the antigen-bindingfunction of an antibody. In accordance with the term “antigen-bindingportion” of an antibody, examples of binding portions include (i) an Fabportion, i.e., a monovalent portion composed of the VL, VH, CL and CH1domains; (ii) an F(ab′)2 portion, i.e., a bivalent portion comprisingtwo Fab portions linked to one another in the hinge region via adisulfide bridge; (iii) an Fd portion composed of the VH and CH1domains; (iv) an Fv portion composed of the FL and VH domains of asingle arm of an antibody; and (v) a dAb portion consisting of a VHdomain or of VH, CH1, CH2, DH3, or VH, CH2, CH3 (dAbs, or single domainantibodies, comprising only VL domains have also been shown tospecifically bind to target eptiopes). Although the two domains of theFv portion, namely VL and VH, are encoded by separate genes, they mayfurther be linked to one another using a synthetic linker, e.g., apoly-G4S amino acid sequence (‘G4S’ disclosed as SEQ ID NO: 29), andrecombinant methods, making it possible to prepare them as a singleprotein chain in which the VL and VH regions combine in order to formmonovalent molecules (known as single chain Fv (ScFv)). The term“antigen-binding portion” of an antibody is also intended to comprisesuch single chain antibodies. Other forms of single chain antibodiessuch as “diabodies” are likewise included here. Diabodies are bivalent,bispecific antibodies in which VH and VL domains are expressed on asingle polypeptide chain, but using a linker which is too short for thetwo domains being able to combine on the same chain, thereby forcingsaid domains to pair with complementary domains of a different chain andto form two antigen-binding sites. An immunoglobulin constant domainrefers to a heavy or light chain constant domain. Human IgG heavy chainand light chain constant domain amino acid sequences are known in theart.

As used herein, the term “antibody reagent” refers to a polypeptide thatincludes at least one immunoglobulin variable domain or immunoglobulinvariable domain sequence and which specifically binds a given antigen.An antibody reagent can comprise an antibody or a polypeptide comprisingan antigen-binding domain of an antibody. In some embodiments of any ofthe aspects, an antibody reagent can comprise a monoclonal antibody or apolypeptide comprising an antigen-binding domain of a monoclonalantibody. For example, an antibody can include a heavy (H) chainvariable region (abbreviated herein as VH), and a light (L) chainvariable region (abbreviated herein as VL). In another example, anantibody includes two heavy (H) chain variable regions and two light (L)chain variable regions. The term “antibody reagent” encompassesantigen-binding fragments of antibodies (e.g., single chain antibodies,Fab and sFab fragments, F(ab′)2, Fd fragments, Fv fragments, scFv, anddomain antibodies (dAb) fragments as well as complete antibodies.

An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM(as well as subtypes and combinations thereof). Antibodies can be fromany source, including mouse, rabbit, pig, rat, and primate (human andnon-human primate) and primatized antibodies. Antibodies also includemidibodies, humanized antibodies, chimeric antibodies, and the like.

Furthermore, an antibody, antigen-binding portion thereof, or CAR asdescribed herein may be part of a larger immunoadhcsion molecule formedby covalent or noncovalent association of said antibody or antibodyportion with one or more further proteins or peptides. Relevant to suchimmunoadhesion molecules are the use of the streptavidin core region inorder to prepare a tetrameric scFv molecule and the use of a cysteinresidue, a marker peptide and a C-terminal polyhistidinyl, e.g.,hexahistidinyl tag (‘hexahistidinyl tag’ disclosed as SEQ ID NO: 40) inorder to produce bivalent and biotinylated scFv molecules.

In some embodiments of any of the aspects, the antibody, antibodyreagent, antigen-binding portion thereof, or CAR described herein can bean immunoglobulin molecule, a monoclonal antibody, a chimeric antibody,a CDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2,a Fv, a disulfide linked Fv, a scFv, a single domain antibody, adiabody, a multispecific antibody, a dual specific antibody, ananti-idiotypic antibody, a bispecific antibody, and a functionallyactive epitope-binding portion thereof.

In some embodiments of any of the aspects, the antibody orantigen-binding portion thereof is a fully human antibody. In someembodiments of any of the aspects, the antibody, antigen-binding portionthereof, is a humanized antibody or antibody reagent. In someembodiments of any of the aspects, the antibody, antigen-binding portionthereof, is a fully humanized antibody or antibody reagent. In someembodiments of any of the aspects, the antibody or antigen-bindingportion thereof, is a chimeric antibody or antibody reagent. In someembodiments of any of the aspects, the antibody, antigen-binding portionthereof, is a recombinant polypeptide. In some embodiments of any of theaspects, the CAR comprises an extracellular domain that binds thetarget, wherein the extracellular domain comprises a humanized orchimeric antibody or antigen-binding portion thereof. In someembodiments of any of the aspects, the CAR comprises an extracellulardomain that binds CHI3L1, wherein the extracellular domain comprises ahumanized or chimeric antibody or antigen-binding portion thereof.

The term “human antibody” refers to antibodies whose variable andconstant regions correspond to or are derived from immunoglobulinsequences of the human germ line, as described, for example, by Kabat etal. (see Kabat, et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242). However, the human antibodies can containamino acid residues not encoded by human germ line immunoglobulinsequences (for example mutations which have been introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs, and in particular in CDR3. Recombinant humanantibodies as described herein have variable regions and may alsocontain constant regions derived from immunoglobulin sequences of thehuman germ line (see Kabat, E. A., et al. (1991) Sequences of Proteinsof Immunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242). According to particularembodiments, however, such recombinant human antibodies are subjected toin-vitro mutagenesis (or to a somatic in-vivo mutagenesis, if an animalis used which is transgenic due to human Ig sequences) so that the aminoacid sequences of the VH and VL regions of the recombinant antibodiesare sequences which although related to or derived from VH and VLsequences of the human germ line, do not naturally exist in vivo withinthe human antibody germ line repertoire. According to particularembodiments, recombinant antibodies of this kind are the result ofselective mutagenesis or back mutation or of both. Preferably,mutagenesis leads to an affinity to the target which is greater, and/oran affinity to non-target structures which is smaller than that of theparent antibody. Generating a humanized antibody from the sequences andinformation provided herein can be practiced by those of ordinary skillin the art without undue experimentation. In one approach, there arefour general steps employed to humanize a monoclonal antibody, see,e.g., U.S. Pat. Nos. 5,585,089; 6,835,823; 6,824,989. These are: (1)determining the nucleotide and predicted amino acid sequence of thestarting antibody light and heavy variable domains; (2) designing thehumanized antibody, i.e., deciding which antibody framework region touse during the humanizing process; (3) the actual humanizingmethodologies/techniques; and (4) the transfection and expression of thehumanized antibody.

Usually the CDR regions in humanized antibodies and human antibodyvariants are substantially identical, and more usually, identical to thecorresponding CDR regions in the mouse or human antibody from which theywere derived. In some embodiments of any of the aspects, it is possibleto make one or more conservative amino acid substitutions of CDRresidues without appreciably affecting the binding affinity of theresulting humanized immunoglobulin or human antibody variant. In someembodiments of any of the aspects, substitutions of CDR regions canenhance binding affinity.

The term “chimeric antibody” refers to antibodies which containsequences for the variable region of the heavy and light chains from onespecies and constant region sequences from another species, such asantibodies having murine heavy and light chain variable regions linkedto human constant regions. Humanized antibodies have variable regionframework residues substantially from a human antibody (termed anacceptor antibody) and complementarity determining regions substantiallyfrom a non-human antibody, e.g., a mouse-antibody, (referred to as thedonor immunoglobulin). The constant region(s), if present, are alsosubstantially or entirely from a human immunoglobulin. The humanvariable domains are usually chosen from human antibodies whoseframework sequences exhibit a high degree of sequence identity with the(murine) variable region domains from which the CDRs were derived. Theheavy and light chain variable region framework residues can besubstantially similar to a region of the same or different humanantibody sequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies or can be consensus sequences ofseveral human antibodies.

In addition, techniques developed for the production of “chimericantibodies” by splicing genes from a mouse, or other species, antibodymolecule of appropriate antigcn specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.The variable segments of chimeric antibodies are typically linked to atleast a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin. Human constant region DNA sequences canbe isolated in accordance with well-known procedures from a variety ofhuman cells, such as immortalized B-cells. The antibody can contain bothlight chain and heavy chain constant regions. The heavy chain constantregion can include CH1, hinge, CH2, CH3, and, sometimes, CH4 regions.For therapeutic purposes, the CH2 domain can be deleted or omitted.

Additionally, and as described herein, a recombinant humanized antibodycan be further optimized to decrease potential immunogenicity, whilemaintaining functional activity, for therapy in humans. In this regard,functional activity means a polypeptide capable of displaying one ormore known functional activities associated with a recombinant antibody,antigen-binding portion thereof, or CAR as described herein. Suchfunctional activities include binding to cancer cells and/or anti-canceractivity. Additionally, a polypeptide having functional activity meansthe polypeptide exhibits activity similar, but not necessarily identicalto, an activity of a reference antibody, antigen-binding portionthereof, or CAR as described herein, including mature forms, as measuredin a particular assay, such as, for example, a biological assay, with orwithout dose dependency. In the case where dose dependency does exist,it need not be identical to that of the reference antibody,antigen-binding portion thereof, or CAR, but rather substantiallysimilar to the dose-dependence in a given activity as compared to thereference antibody, antigen-binding portion thereof, or CAR as describedherein (i.e., the candidate polypeptide will exhibit greater activity,or not more than about 25-fold less, about 10-fold less, or about 3-foldless activity relative to the antibodies, antigen-binding portions,and/or CARS described herein).

In some embodiments of any of the aspects, the antibody reagents (e.g.,antibodies or CARS) described herein are not naturally-occurringbiomolecules. For example, a murine antibody raised against an antigenof human origin would not occur in nature absent human intervention andmanipulation, e.g., manufacturing steps carried out by a human. Chimericantibodies are also not naturally-occurring biomolecules, e.g., in thatthey comprise sequences obtained from multiple species and assembledinto a recombinant molecule. In certain particular embodiments, thehuman antibody reagents described herein are not naturally-occurringbiomolecules, e.g., fully human antibodies directed against a humanantigen would be subject to negative selection in nature and are notnaturally found in the human body.

In some embodiments of any of the aspects, the antibody, antibodyreagent, antigen-binding portion thereof, and/or CAR is an isolatedpolypeptide. In some embodiments of any of the aspects, the antibody,antibody reagent, antigen-binding portion thereof, and/or CAR is apurified polypeptide. In some embodiments of any of the aspects, theantibody, antibody reagent, antigen-binding portion thereof, and/or CARis an engineered polypeptide.

In one aspect of any of the embodiments, described herein is anantibody, antigen-binding fragment thereof, antigen reagent or chimaericantigen receptor (CAR) which is an inhibitor of CHI3L1 specificallybinds a CHI3L1 polypeptide. In some embodiments of any of the aspects,the antibody, antigen-binding fragment thereof, antigen reagent or CARwhich is an inhibitor of CHI3L1 comprises at least one heavy or lightchain complementarity determining region (CDR) selected from the groupconsisting of:

-   -   (a) a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 4;    -   (b) a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 5;    -   (c) a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 6;    -   (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 1;    -   (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 2; and    -   (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 3; or        a conservative substitution variant of one or more of (a)-(f).        In some embodiments of any of the aspects, the antibody,        antigen-binding fragment thereof, antigen reagent or CAR        comprises at least one heavy or light chain complementarity        determining region (CDR) selected from the group consisting of:    -   (a) a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 4;    -   (b) a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 5;    -   (c) a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 6;    -   (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 1;    -   (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 2; and    -   (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 3.

In one aspect of any of the embodiments, described herein is anantibody, antigen-binding fragment thereof, antigen reagent or chimaericantigen receptor (CAR) which is an inhibitor of CHI3L1 specificallybinds a CHI3L1 polypeptide. In some embodiments of any of the aspects,the antibody, antigen-binding fragment thereof, antigen reagent or CARwhich is an inhibitor of CHI3L1 comprises a heavy chain CDR3 having theamino acid sequence of SEQ ID NO: 3. In some embodiments of any of theaspects, the antibody, antigen-binding fragment thereof, antigen reagentor CAR comprises a heavy chain CDR1 having the amino acid sequence ofSEQ ID NO: 1; a heavy chain CDR2 having the amino acid sequence of SEQID NO: 2; and a heavy chain CDR3 having the amino acid sequence of SEQID NO: 3.

In some embodiments of any of the aspects, the antibody, antibodyreagent, antigen-binding portion thereof, or CAR that specifically bindsan CHI3L1 polypeptide binds specifically to an epitope selected from SEQID NOs: 13-24. In some embodiments of any of the aspects, the antibody,antibody reagent, antigen-binding portion thereof, or CAR thatspecifically binds an CHI3L1 polypeptide binds specifically to theepitope of SEQ ID NO: 13.

In some embodiments of any of the aspects, the antibody, antigen-bindingfragment thereof, antigen reagent or CAR which is an inhibitor of CHI3L1comprises heavy chain CDRs having the amino acid sequences of SEQ IDNOs: 1-3. In some embodiments of any of the aspects, the antibody,antigen-binding fragment thereof, antigen reagent or CAR comprises heavychain CDRs having the amino acid sequences of SEQ ID NOs: 1-3 or aconservative substitution variant of such amino acid sequence.

In some embodiments of any of the aspects, the antibody, antigen-bindingfragment thereof, antigen reagent or CAR which is an inhibitor of CHI3L1comprises light chain CDRs having the amino acid sequences of SEQ IDNOs: 4-6. In some embodiments of any of the aspects, the antibody,antigen-binding fragment thereof, antigen reagent or CAR which is aninhibitor of CHI3L1 comprises light chain CDRs having the amino acidsequences of SEQ ID NOs: 4-6 or a conservative substitution variant ofsuch amino acid sequence.

In some embodiments of any of the aspects, the antibody, antigen-bindingfragment thereof, antigen reagent or CAR which is an inhibitor of CHI3L1comprises the heavy or light chain complementarity determining region(CDR)s as follows:

-   -   (a) a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 4;    -   (b) a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 5;    -   (c) a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 6;    -   (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 1;    -   (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 2; and    -   (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 3.        In some embodiments of any of the aspects, the antibody,        antigen-binding fragment thereof, antigen reagent or CAR which        is an inhibitor of CHI3L1 comprises the heavy or light chain        complementarity determining region (CDR)s as follows:    -   (a) a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 4;    -   (b) a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 5;    -   (c) a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 6;    -   (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 1;    -   (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 2; and    -   (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 3; or a conservative substitution variant of the amino acid        sequence of any of (a)-(f). In some embodiments of any of the        aspects, the antibody, antibody reagent, antigen-binding portion        thereof, or CAR that specifically binds an CHI3L1 polypeptide        binds specifically to an epitope selected from SEQ ID NOs:        13-24. In some embodiments of any of the aspects, the antibody,        antibody reagent, antigen-binding portion thereof, or CAR that        specifically binds an CHI3L1 polypeptide binds specifically to        the epitope of SEQ ID NO: 13.

In some embodiments of any of the aspects, the antibody, antibodyreagent, antigen-binding portion thereof, or CAR which is an inhibitorof CHI3L1 can comprise one or more CDRs (e.g., one CDR, two CDRs, threeCDRs, four CDRs, five CDRs, or six CDRs) having the sequence of a CDRselected from SEQ ID NOs: 1-6. In some embodiments of any of theaspects, the antibody, antibody reagent, antigen-binding portionthereof, or CAR which is an inhibitor of CHI3L1 can comprise CDRs havingthe sequence of the CDRs of SEQ ID NOs: 1-6.

In one aspect of any of the embodiments, described herein is anantibody, antibody reagent, antigen-binding portion thereof, or CARwhich is an inhibitor of CHI3L1 that specifically binds an CHI3L1polypeptide, and can compete for binding of CHI3L1 with an antibodycomprising light chain CDRs having the amino acid sequences of SEQ IDNOs: 4-6 and heavy chain CDRs having the amino acid sequences of SEQ IDNOs: 1-3.

In some embodiments of any of the aspects, the antibody, antibodyreagent, antigen-binding portion thereof, or CAR can comprise a heavychain sequence having the amino acid sequence of SEQ ID NO: 36 and/or alight chain sequence having the amino acid sequence of SEQ ID NO: 38.

FRG Heavy chain sequence  (SEQ ID NO: 36)QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLRNEDMSTYFCARLG YGKFYVMDYWGQGTSVTVSSFRG Heavy chain nucleotide sequence  (SEQ ID NO: 37)CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAATACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAGAAATGAGGACATGTCTACATATTTCTGTGCAAGATTGGGATATGGTAAATTCTATGTTATGGACTACTGGGGTCAGGGAACGTCAGTCAC CGTCTCCTCAFRG Light chain sequence  (SEQ ID NO: 38)DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKWYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVTWT FGGGTKLEIKFRG Heavy chain nucleotide sequence  (SEQ ID NO: 39)GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTACGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

In some embodiments of any of the aspects, the antibody, antibodyreagent, antigen-binding portion thereof, or CAR which is an inhibitorof CHI3L1 that specifically binds an CHI3L1 polypeptide bindsspecifically to an epitope selected from SEQ ID NOs: 13-24. In someembodiments of any of the aspects, the antibody, antibody reagent,antigen-binding portion thereof, or CAR that specifically binds anCHI3L1 polypeptide binds specifically to the epitope of SEQ ID NO: 13.

Additional anti-CHI3L1 reagents are known in the art which can be usedin the methods described herein, including Cat Nos. PA5-46996,PA5-43746, PA5-37357, and PA5-47363 from Invitrogen (Invitrogen LifeTech, Carlsbad, Calif.) and antibody reagents comprising one or more(e.g., all six) of the CDRs of those antibodies.

In some embodiments of any of the aspects, the antibody, antibodyreagent, antigen-binding portion thereof, and/or CAR as described hereincan be a variant of a sequence described herein, e.g., a conservativesubstitution variant of an antibody polypeptide. In some embodiments ofany of the aspects, the variant is a conservatively modified variant.Conservative substitution variants can be obtained by mutations ofnative nucleotide sequences, for example. A “variant,” as referred toherein, is a polypeptide substantially homologous to a native orreference polypeptide, but which has an amino acid sequence differentfrom that of the native or reference polypeptide because of one or aplurality of deletions, insertions or substitutions. Variantpolypeptide-encoding DNA sequences encompass sequences that comprise oneor more additions, deletions, or substitutions of nucleotides whencompared to a native or reference DNA sequence, but that encode avariant protein or portion thereof that retains activity, e.g.,antigen-specific binding activity for the relevant target polypeptide,e.g., CHI3L1. A wide variety of PCR-based site-specific mutagenesisapproaches are also known in the art and can be applied by theordinarily skilled artisan.

One of skill will recognize that individual substitutions, deletions oradditions to a nucleic acid, peptide, polypeptide, or protein sequencewhich alters a single amino acid or a small percentage of amino acids inthe encoded sequence is a “conservatively modified variant” where thealteration results in the substitution of an amino acid with achemically similar amino acid and retain the ability to specificallybind the target antigen (e.g., CHI3L1). Such conservatively modifiedvariants are in addition to and do not exclude polymorphic variants,interspecies homologs, and alleles consistent with the disclosure.

Examples of substitution variants include conservative substitution ofamino acids, e.g., in a V_(H) or V_(L), domain, that do not alter thesequence of a CDR. A conservative substitution in a sequence notcomprised by a CDR can be a substitution relative to a wild-type ornaturally-occurring sequence, e.g., human or murine framework and/orconstant regions of an antibody sequence. In some embodiments of any ofthe aspects, a conservatively modified variant of an antibody reagentcan comprise alterations other than in the CDRs, e.g., a conservativelymodified variant of an antibody, antibody reagent, antigen-bindingportion thereof, or CAR which is an inhibitor of CHI3L1 can compriseCDRs having the sequence of one or more of SEQ ID NOs 1-6. In someembodiments of any of the aspects, a conservatively modified variant ofan antibody, antibody reagent, antigen-binding portion thereof, or CARwhich is an inhibitor of CHI3L1 can comprise CDRs having the sequencesof SEQ ID NOs: 1-6.

A given amino acid can be replaced by a residue having similarphysiochemical characteristics, e.g., substituting one aliphatic residuefor another (such as Ile, Val, Leu, or Ala for one another), orsubstitution of one polar residue for another (such as between Lys andArg; Glu and Asp; or Gln and Asn). Other such conservativesubstitutions, e.g., substitutions of entire regions having similarhydrophobicity characteristics, are well known. Polypeptides comprisingconservative amino acid substitutions can be tested in any one of theassays described herein to confirm that a desired activity, e.g.,antigen-binding activity and specificity of a native or referencepolypeptide is retained.

Amino acids can be grouped according to similarities in the propertiesof their side chains (in A. L. Lehninger, in Biochemistry, second ed.,pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A),Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2)uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N),Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His(H). Alternatively, naturally occurring residues can be divided intogroups based on common side-chain properties: (1) hydrophobic:Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser,Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5)residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp,Tyr, Phc. Non-conservative substitutions will entail exchanging a memberof one of these classes for another class. Particular conservativesubstitutions include, for example; Ala into Gly or into Ser; Arg intoLys; Asn into Gln or into H is; Asp into Glu; Cys into Ser; Gln intoAsn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln;Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, intoGln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, intoLeu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp;and/or Phe into Val, into Ile or into Leu.

A variant amino acid or DNA sequence preferably is at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or more, identicalto a native or reference sequence. The degree of homology (percentidentity) between a native and a mutant sequence can be determined, forexample, by comparing the two sequences using freely available computerprograms commonly employed for this purpose on the world wide web (e.g.,BLASTp or BLASTn with default settings).

Alterations of the native amino acid sequence can be accomplished by anyof a number of techniques known to one of skill in the art. Mutationscan be introduced, for example, at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered nucleotide sequencehaving particular codons altered according to the substitution,deletion, or insertion required.

Any cysteine residue not involved in maintaining the proper conformationof the polypeptide also can be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, a cysteine bond(s) can be added to thepolypeptide to improve its stability or facilitate oligomerization.

In particular embodiments wherein an antibody, antigen-binding portionthereof, or CAR which is an inhibitor of CHI3L1 comprises at least oneCDR which is not identical to the sequence of SEQ ID NOs: 1-6, the aminoacid sequence of that at least one CDR can be selected by methods wellknown to one of skill in the art. For example, Fujii, 2004, “Antibodyaffinity maturation by random mutagenesis” in Methods in MolecularBiology: Antibody Engineering 248: 345-349 (incorporated by referenceherein in its entirety), particularly at FIG. 2 and Section 3.3,describes methods of generating a library for any CDR of interest. Thisallows one of ordinary skill in the art to identify alternative CDRs,including conservative substitution variants of the specific CDRsequences described herein, which, when present in an antibody orantigen-binding portion thereof as described herein, will result in anantigen or antigen-binding portion thereof which will bind a cancer cellsurface antigen. The method described in Fujii et al. also permits oneof ordinary skill in the art to screen for a light chain sequence whichwill give the desired binding behavior when combined with a known heavychain fragment and vice versa.

In some embodiments of any of the aspects, a CAR which is an inhibitorof CHI3L1 comprises an extracellular domain comprising an anti-CHI3L1antibody or antigen-binding portion thereof that binds one or moreepitopes of a CHI3L1 polypeptide; a transmembrane domain, one or moreintracellular co-stimulatory signaling domains, and a primary signalingdomain. Exemplary anti-CHI3L1 antibodies and antigen-binding portionsthereof, as well as exemplary epitopes, are described elsewhere herein

As used herein, “chimeric antigen receptor” or “CAR” refers to anartificially constructed hybrid polypeptide comprising anantigen-binding domain (e.g., an antigen-binding portion of an antibody(e.g., a scFV)), a transmembrane domain, and a T-cell signaling and/orT-cell activation domain. CARS have the ability to redirect T-cellspecificity and reactivity toward a selected target in anon-MHC-restricted manner, exploiting the antigen-binding properties ofmonoclonal antibodies. The non-MHC-restricted antigen recognition givesT-cells expressing CARS the ability to recognize an antigen independentof antigen processing, thus bypassing a major mechanism of tumor escape.Moreover, when expressed in T-cells, CARs advantageously do not dimerizewith endogenous T-cell receptor (TCR) alpha and beta chains. Mostcommonly, the CAR's extracellular binding domain is composed of a singlechain variable fragment (scFv) derived from fusing the variable heavyand light regions of a murine or humanized monoclonal antibody.Alternatively, scFvs may be used that are derived from Fab's (instead offrom an antibody, e.g., obtained from Fab libraries), in variousembodiments, this scFv is fused to a transmembrane domain and then to anintracellular signaling domain. “First-generation” CARs include thosethat solely provide CD3zeta (CD3ζ) signals upon antigen binding,“Second-generation” CARS include those that provide both costimulation(e.g., CD28 or CD 137) and activation (CD3ζ). “Third-generation” CARsinclude those that provide multiple costimulatory (e.g., CD28 and CD137) domains and activation domains (e.g., CD3ζ). In variousembodiments, the CAR is selected to have high affinity or avidity forthe antigen. Further discussion of CARs can be found, e.g., in Maus etal. Blood 2014 123:2624-35; Reardon et al. Neuro-Oncology 201416:1441-1458; Hoyos et al. Haematologica 2012 97:1622; Byrd et al. JClin Oncol 2014 32:3039-47; Maher et al. Cancer Res 2009 69:4559-4562;and Tamada et al. Clin Cancer Res 2012 18:6436-6445; each of which isincorporated by reference herein in its entirety.

In some embodiments of any of the aspects, a CAR which is an inhibitorof CHI3L1 comprises an extracellular binding domain that comprises ahumanized CHI3L1-specific binding domain; a transmembrane domain; one ormore intracellular co-stimulatory signaling domains; and a primarysignaling domain. As used herein, the terms, “binding domain,”“extracellular domain,” “extracellular binding domain,”“antigen-specific binding domain,” and “extracellular antigen specificbinding domain,” are used interchangeably and provide a CAR with theability to specifically bind to the target antigen of interest, e.g.,CHI3L1. The binding domain may be derived either from a natural,synthetic, semi-synthetic, or recombinant source.

In some embodiments of any of the aspects, the CARs contemplated hereinmay comprise linker residues between the various domains, e.g., addedfor appropriate spacing and conformation of the molecule. In particularembodiments the linker is a variable region linking sequence. A“variable region linking sequence,” is an amino acid sequence thatconnects the VH and VL domains and provides a spacer function compatiblewith interaction of the two sub-binding domains so that the resultingpolypeptide retains a specific binding affinity to the same targetmolecule as an antibody that comprises the same light and heavy chainvariable regions. CARS contemplated herein, can comprise one, two,three, four, or five or more linkers. In particular embodiments, thelength of a linker is about 1 to about 25 amino acids, about 5 to about20 amino acids, or about 10 to about 20 amino acids, or any interveninglength of amino acids. In some embodiments of any of the aspects, thelinker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, or more amino acids long.

In particular embodiments, the binding domain of the CAR is followed byone or more “spacer domains,” which refers to the region that moves theantigen binding domain away from the effector cell surface to enableproper cell/cell contact, antigen binding and activation. The hingedomain may be derived either from a natural, synthetic, semi-synthetic,or recombinant source. In certain embodiments, a spacer domain is aportion of an immunoglobulin, including, but not limited to, one or moreheavy chain constant regions, e.g., CH2 and CH3. The spacer domain caninclude the amino acid sequence of a naturally occurring immunoglobulinhinge region or an altered immunoglobulin hinge region.

The binding domain of the CAR is generally followed by one or more“hinge domains,” which plays a role in positioning the antigen bindingdomain away from the effector cell surface to enable proper cell/cellcontact, antigen binding and activation. A CAR generally comprises oneor more hinge domains between the binding domain and the transmembranedomain (TM). The hinge domain may be derived either from a natural,synthetic, semi-synthetic, or recombinant source. The hinge domain caninclude the amino acid sequence of a naturally occurring immunoglobulinhinge region or an altered immunoglobulin hinge region. Illustrativehinge domains suitable for use in the CARS described herein include thehinge region derived from the extracellular regions of type 1 membraneproteins such as CD8α, CD4, CD28 and CD7, which may be wild-type hingeregions from these molecules or may be altered. In another embodiment,the hinge domain comprises a CD8α hinge region.

The “transmembrane domain” is the portion of the CAR that fuses theextracellular binding portion and intracellular signaling domain andanchors the CAR to the plasma membrane of the immune effector cell. TheTM domain may be derived either from a natural, synthetic,semi-synthetic, or recombinant source. The TM domain may be derived from(i.e., comprise at least the transmembrane region(s) of) the alpha, betaor zeta chain of the T-cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9,CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134,CD137, CD152, CD 154, and PD1.

In some embodiments of any of the aspects, CARS contemplated hereincomprise an intracellular signaling domain. An “intracellular signalingdomain,” refers to the part of a CAR that participates in transducingthe message of effective CAR binding to a target antigen into theinterior of the immune effector cell to elicit effector cell function,e.g., activation, cytokine production, proliferation and cytotoxicactivity, including the release of cytotoxic factors to the CAR-boundtarget cell, or other cellular responses elicited with antigen bindingto the extracellular CAR domain. In some embodiments of any of theaspects, a CAR contemplated herein comprises an intracellular signalingdomain that comprises one or more “co-stimulatory signaling domain” anda “primary signaling domain.”

Primary signaling domains regulate primary activation of the TCR complexeither in a stimulatory way, or in an inhibitory way. Primary signalingdomains that act in a stimulatory manner may contain signaling motifswhich are known as immunoreceptor tyrosine-based activation motifs orITAMs. Illustrative examples of 1TAM containing primary signalingdomains that are of particular use in the invention include thosederived from TCR, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a,CD79b, and CD66d.

As used herein, the term, “co-stimulatory signaling domain,” or“co-stimulatory domain”, refers to an intracellular signaling domain ofa co-stimulatory molecule. Co-stimulatory molecules are cell surfacemolecules other than antigen receptors or Fc receptors that provide asecond signal required for efficient activation and function of Tlymphocytes upon binding to antigen. Illustrative examples of suchco-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30,CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1),CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1),CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In oneembodiment, a CAR comprises one or more co-stimulatory signaling domainsselected from the group consisting of CD28, CD137, and CD134, and a CD3ζprimary signaling domain.

In some embodiments of any of the aspects, the inhibitor of CHI3L1signaling described herein is an inhibitor of a CHI3L1 receptor. CHI3L1receptors include CRTH2 and the heterodimer receptor formed by IL13Rα2and TMEM219.

As used herein, “CRTH2,” “chemoattractant homologous receptor expressedon Th2 cells,” “CD294,” or “GPR44” refers to a G protein-coupledreceptor activated by prostaglandin D₂ and/or CHI3L1. Activation ofCRTH2 by prostaglandin D₂ induces the chemotaxis of Th2 lymphocytes andeosinophils, even in the absence of allergen or co-stimulation.Activation of CRTH2 by CHI3L1 can induce fibroproliferation. In someembodiments of any of the aspects, CRTH2 activity can be measured bymeasuring CHI3L1-induced fibroproliferation. In some embodiments of anyof the aspects, increased CRTH2 activity can be indicated by increasedM2 macrophage differentiation. The sequences of CRTH2 expressionproducts for a number of species are known, e.g., human CRTH2 (NCBI GeneID: 11251) mRNA (NCBI Ref Seq: NM_004778) and polypeptide (NCBI Ref Seq:NP_004769).

As used herein, “IL-13Rα2,” “Interleukin-13 receptor subunit alpha-2,”or “CD213A2” refers to a membrane bound protein that forms a heterodimerwith TMEM219 to form a CHI3L1 receptor. IL13Rα2 also binds to IL-13 withvery high affinity, and negatively regulates both IL-13 and TL-4.IL-13Rα2 competes with the IL-13 receptor comprising IL-13Rα1 andIL4Ralpha for binding of IL-13. Sequences for IL-13Rα2 expressionproducts are known for a number of species, e.g., human IL-13Rα2 (NCBIGene ID: 3598) mRNA (NCBI Ref Seq: NM_000640) and polypeptide (NCBI RefSeq: NP_000631).

As used herein, “TMEM219” or “transmembrane protein 219” refers to amembrane bound protein that forms a heterodimer with IL-13Rα2 to form aCHI3L1 receptor. Sequences for TMEM219 expression products are known fora number of species, e.g., human TMEM219 (NCBI Gene ID: 124446) mRNA(NCBI Ref Seq: NM_001083613.1 and NM_194280.3) and polypeptide (NCBI RefSeq: NP_001077082.1 and NP_919256.1).

In some embodiments of any of the aspects, inhibitors of CHI3L1receptors can be inhibitory antibodies, antibody reagents,antigen-binding fragments thereof, or CARs that bind specifically to thereceptor and/or receptor subunit.

Exemplary antibodies, antibody reagents, and/or antigen-bindingfragments thereof, which are inhibitors of CHI3L1 receptors are known inthe art and can include Cat Nos. PA5-46996, PA5-43746, PA5-37357, andPA5-47363 from Invitrogen (Invitrogen Life Tech, Carlsbad, Calif.)

In some embodiments of any of the aspects, inhibitors (e.g., of Sema7A,β1-integrin, an immune checkpoint protein, CHI3L1 and/or CHI3L1receptors) can be inhibitory nucleic acid molecules. In some embodimentsof any of the aspects, an inhibitory nucleic acid molecule can be amiRNA, iRNA, amiRNA, RNAi molecule or the like.

In some embodiments of any of the aspects, multiple inhibitors oragonists of a given target can be used in the methods and compositionsdescribed herein, e.g., two or more inhibitors that target the sametarget (e.g., CHI3L1), or at least one inhibitor that targets a firsttarget (e.g., CHI3L1) and at least one inhibitor that targets at leastone additional target (e.g., one or more CHI3L1 receptors). In someembodiments of any of the aspects, multiple inhibitors of CHI3L1 can beused in the methods and compositions described herein, e.g., two or moreinhibitors that target the same target (e.g., CHI3L1), or at least oneinhibitor that targets a first target (e.g., CHI3L1) and at least oneinhibitor that targets at least one additional target (e.g., one or moreCHI3L1 receptors).

As used herein, “semaphorin 7A” or “Sema7A” (also known as CD108) refersto a membrane-bound member of the semaphorin family and which isexpressed on activated lymphocytes and erythrocytes. The sequences ofSema7A expression products are known for a number of species, e.g.,human Sema7A (NCBI Gene ID No: 8482) mRNA (SEQ ID NO: 41; NCBI Ref Seq:NM_001146029.2, SEQ ID NO: 42 NCBI Ref Seq: 001146030.2 and SEQ ID NO:43 NCBI Ref Seq: NM_003612.4) and polypeptide (SEQ ID NO: 44 NCBI RefSeq: NP_001139501.1; SEQ ID NO: 45 NCBI Ref Seq: NP_001139502.1 and SEQID NO: 46 NCBI Ref Seq: NP_003603.1).

Inhibitors of Sema7A are known in the art and can include anti-Sema7Aantibody reagents or Sema7A inhibitory nucleic acid molecules.Exemplary, non-limiting examples of anati-Sema7A antibodies can includeAF1835 from R and D Systems of Minneapolis Minn.; ab8222 (MEM-150),ab23578, ab217628, ab90242 (MM0537-99D22), or ab133803 from AbCam ofCambridge, Mass.; PAS-50753, PAS-28971, or PAS-47246 from ThermoFisherScientific of Waltham, Mass.; C-6 (sc-374432) from Santa CruzBiotechnology of Dallas, Tex.; and 191-205 (SAB1105046) or 376-390(SAB1105047) from Sigma Aldrich of St. Louis, Mo.;

As used herein, “beta 1 integrin” or “β1-integrin” (also known as ITGB1or CD29) refers to cell surface receptor that interacts with a number ofdifferent partners to form receptor complexes. The sequences ofβ1-integrin expression products are known for a number of species, e.g.,human β1-integrin (NCBI Gene ID No: 3688) mRNA (SEQ ID NO: 47; NCBI RefSeq: NM_002211.3, SEQ ID NO: 48 NCBI Ref Seq: NM_033668.2 and SEQ ID NO:49* NCBI Ref Seq: NM_133376.2) and polypeptide (SEQ ID NO: 50 NCBI RefSeq: NP_002202.2; SEQ ID NO: 51 NCBI Ref Seq: NP_391988.1 and SEQ ID NO:52 NCBI Ref Seq: NP_596867.1).

Inhibitors of β1-integrin are known in the art and can includeanti-β1-integrin antibody reagents or β1-integrin inhibitory nucleicacid molecules. Exemplary, non-limiting examples of anti-β1-integrinantibodies can include P5D2 (ab24693), EPR16895 (ab179471), EP1041Y(ab52971), 12G10 (ab30394), ab183666, JB1B (2630388), and BV7 (ab7168)from AbCam of Cambridge, Mass.; 3B6 (MA5-17103), PA5-29606, 44-872G,HMb1-1 (11-0291-82), TS2/16 (11-0299-42), PA5-78028, MEM101A (CD2920),4B7R (MA5-13658), 2B1 (MA1-06906), KMI6 (14-0292-82) from ThermoFisherScientific of Waltham, Mass.; M106 (sc-8978) or A-4 (sc-374429) fromSanta Cruz Biotechnology of Dallas, Tex.; and MABT409 or AB1952 fromMillipore Sigma of Burlington, Mass.

It is further contemplated herein that certain embodiments of any of theaspects can relate to use or administration of both an inhibitor ofSema7A and of β1-integrin for particularly efficacious results. In someembodiments, a single bivalent antibody reagent is an inhibitor of bothSema7A and β1-integrin, e.g., one specificity is for Sema7A and thesecond specificity is for β1-integrin.

As used herein, “Plexin C1” or “PLXNC1” (also known as CD232 or VESPR)refers to a member of the plexin family of semaphorin receptors thatbinds Sema7A as well as a number of viral semaphorins. The sequences ofPlexin C1 expression products are known for a number of species, e.g.,human Plexin C1 (NCBI Gene ID No: 10154) mRNA (SEQ ID NO: 53; NCBI RefSeq: NM_005761.2) and polypeptide (SEQ ID NO: 54 NCBI Ref Seq:NP_005752.1).

Agonists of Plexin C1 are known in the art and can include Plexin C1polypeptides, nucleic acids encoding Plexin C1, or nucleic acidsencoding Growth Arrest Specific 5 (GASS, NCBI Gene ID: 60674) which is aknown agonist of Plexin C1 (e.g., see Zhao et al. Mol Ther 23:189-19114(2015); which is incorporated by reference herein in its entirety).

As used herein, the term “agonist” refers to an agent which increasesthe expression and/or activity of the target by at least 10% or more,e.g. by 10% or more, 50% or more, 100% or more, 200% or more, 500% ormore, or 1000% or more. The efficacy of an agonist of, for example,Plexin C1, e.g. its ability to increase the level and/or activity of thetarget can be determined, e.g. by measuring the level of an expressionproduct of the target and/or the activity of the target. Methods formeasuring the level of a given mRNA and/or polypeptide are known to oneof skill in the art, e.g. RTPCR with primers can be used to determinethe level of RNA, and Western blotting with an antibody can be used todetermine the level of a polypeptide. Suitable primers for a giventarget are readily identified by one of skill in the art, e.g., usingsoftware widely available for this purpose (e.g., Primer3 or PrimerBank,which are both available on the world wide web). Non-limiting examplesof antibodies to, e.g., PlexinC1, are commercially available, e.g., Cat.No. ab116070 from AbCam (Cambridge, Mass.). Assays for measuring theactivity of the targets described herein are provided elsewhere herein.In some embodiments of any of the aspects, an agonist of a givenpolypeptide can be the polypeptide, a nucleic acid encoding thepolypeptide, or a small molecule.

Non-limiting examples of agonists of a given polypeptide target, caninclude the target polypeptides or variants or functional fragmentsthereof and nucleic acids encoding the polypeptide or variants orfunctional fragments thereof. In some embodiments of any of the aspects,the agonist of a given target, is a polypeptide of that target orvariants or functional fragment thereof and/or a nucleic acid encodingthe polypeptide or variant or functional fragment thereof. In someembodiments of any of the aspects, the polypeptide agonist can be anengineered and/or recombinant polypeptide. In some embodiments of any ofthe aspects, the polypeptide agonist can be a nucleic acid encoding apolypeptide, e.g. a functional fragment thereof. In some embodiments ofany of the aspects, the nucleic acid can be comprised by a vector.

In some embodiments of any of the aspects, a polypeptide agonist cancomprise one of the sequences provided below herein for each target. Insome embodiments of any of the aspects, a polypeptide agonist canconsist essentially of one of the sequences provided below herein foreach target. In some embodiments of any of the aspects, a polypeptideagonist can consist of one of the sequences provided below herein foreach target. In some embodiments of any of the aspects, an agonist cancomprise a nucleic acid encoding one of the sequences provided belowherein for each target. In some embodiments of any of the aspects, anagonist can be a polypeptide comprising a reference/wild-type sequenceprovided herein with at least 80%, at least 85%, at least 90%, at least95%, or at least 98% identity to the reference/wild-type sequence andwhich retains the activity of the reference/wild-type sequence.

In some embodiments of any of the aspects, the agonist an exogenouspolypeptide. In some embodiments of any of the aspects, the targetcell(s) and/or subject is contacted with and/or administered exogenouspolypeptide, e.g., the polypeptide is produced in vitro and/orsynthesized and purified polypeptide is provided to the target cell(s)and/or subject.

In some embodiments of any of the aspects, the agonist can be a nucleicacid encoding a polypeptide (or a variant or functional fragmentthereof) and/or a vector comprising a nucleic acid encoding apolypeptide (or a variant or functional fragment thereof). A nucleicacid encoding a polypeptide can be, e.g., an RNA molecule, a plasmid,and/or an expression vector. In some embodiments of any of the aspects,the nucleic acid encoding a polypeptide can be an mRNA. In someembodiments of any of the aspects, the nucleic acid encoding apolypeptide can be a modified mRNA. In some embodiments of any of theaspects, the agonist can be a nucleic acid encoding a polypeptide, e.g.,exogenous and/or ectopic polypeptide. In some embodiments of any of theaspects, the target cell(s) and/or subject is contacted with and/oradministered the nucleic acid encoding exogenous and/or ectopicpolypeptide, e.g., the nucleic acid is transcribed and/or translatedafter the contacting or administering step to provide exogenous and/orectopic to the target cell(s) and/or subject.

The immune system has multiple inhibitory pathways that are critical formaintaining self-tolerance and modulating immune responses. For example,in T-cells, the amplitude and quality of response is initiated throughantigen recognition by the T-cell receptor and is regulated by immunecheckpoint proteins that balance co-stimulatory and inhibitory signals.In some embodiments of any of the aspects, a subject or patient istreated with at least one inhibitor of an immune checkpoint protein. Asused herein, “immune checkpoint protein” refers to a protein which, whenactive, exhibits an inhibitory effect on immune activity, e.g., T cellactivity. Exemplary immune checkpoint proteins can include PD-1 (e.g.,NCBI Gene ID: 5133); PD-L1 (e.g., NCBI Gene ID: 29126); PD-L2 (e.g.,NCBI Gene ID: 80380); TIM-3 (e.g., NCBI Gene ID: 84868); CTLA4 (e.g.,NCBI Gene ID: 1493); TIGIT (e.g., NCBI Gene ID: 201633); KIR (e.g., NCBIGene ID: 3811); LAG3 (e.g., NCBI Gene ID: 3902); DD1-a (e.g., NCBI GeneID: 64115); A2AR (e.g., NCBI Gene ID: 135); B7-H3 (e.g., NCBI Gene ID:80381); B7-H4 (e.g., NCBI Gene ID: 79679); BTLA (e.g., NCBI Gene ID:151888); IDO (e.g., NCBI Gene ID: 3620); TDO (e.g., NCBI Gene ID: 6999);HVEM (e.g., NCBI Gene ID: 8764); GALS (e.g., NCBI Gene ID: 3965); 2B4(belongs to the CD2 family of molecules and is expressed on all NK, γδ,and memory CD8+(αβ) T cells) (e.g., NCBI Gene ID: 51744); CD160 (alsoreferred to as BY55) (e.g., NCBI Gene ID: 11126); and various B-7 familyligands. B7 family ligands include, but are not limited to, B7-1, B7-2,B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7.

Programmed cell death 1 (PD-1), limits the activity of T cells inperipheral tissues at the time of an inflammatory response to infectionand limits autoimmunity. PD-1 blockade in vitro enhances T-cellproliferation and cytokine production in response to a challenge byspecific antigen targets or by allogeneic cells in mixed lymphocytereactions. A strong correlation between PD-1 expression and response wasshown with blockade of PD-1 (Pardoll, Nature Reviews Cancer, 12:252-264, 2012). PD-1 blockade can be accomplished by a variety ofmechanisms including antibodies that bind PD-1 or its ligand, PD-L1.Examples of PD-1 and PD-L1 blockers are described in U.S. Pat. Nos.7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT PublishedPatent Application Nos: WO03042402, WO2008156712, WO2010089411,WO2010036959, WO2011066342, WO2011159877, WO2011082400, andWO2011161699; which are incorporated by reference herein in theirentireties. In certain embodiments the PD-1 inhibitors includeanti-PD-L1 antibodies. In certain other embodiments the PD-1 inhibitorsinclude anti-PD-1 antibodies and similar binding proteins such asnivolumab (MDX 1106, BMS 936558, ONO 4538), a fully human IgG4 antibodythat binds to and blocks the activation of PD-1 by its ligands PD-L1 andPD-L2; lambrolizumab (MK-3475 or SCH 900475), a humanized monoclonalIgG4 antibody against PD-1; CT-011 a humanized antibody that binds PD-1;AMP-224, a fusion protein of B7-DC; an antibody Fc portion; BMS-936559(MDX-1105-01) for PD-L1 (B7-H1) blockade. Also specifically contemplatedherein are agents that disrupt or block the interaction between PD-1 andPD-L1, such as a high affinity PD-L1 antagonist.

Cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) is an immunecheckpoint protein that downregulates pathways of T-cell activation(Fong et al., Cancer Res. 69(2):609-615, 2009; Weber Cancer Immunol.Immunother, 58:823-830, 2009). Blockade of CTLA-4 has been shown toaugment T-cell activation and proliferation. Inhibitors of CTLA-4include anti-CTLA-4 antibodies. Anti-CTLA-4 antibodies bind to CTLA-4and block the interaction of CTLA-4 with its ligands CD80/CD86 expressedon antigen presenting cells, thereby blocking the negative downregulation of the immune responses elicited by the interaction of thesemolecules. Examples of anti-CTLA-4 antibodies are described in U.S. Pat.Nos. 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; 6,682,736;6,984,720; and 7,605,238; which are incorporated by reference herein intheir entireties. One anti-CDLA-4 antibody is tremelimumab,(ticilimumab, CP-675,206). In one embodiment, the anti-CTLA-4 antibodyis ipilimumab (also known as 10D1, MDX-D010) a fully human monoclonalIgG antibody that binds to CTLA-4. Ipilimumab is marketed under the nameYERVOY™ and has been approved for the treatment of unresectable ormetastatic melanoma.

Additional anti-CTLA4 antagonists include, but are not limited to, thefollowing: any inhibitor that is capable of disrupting the ability ofCD28 antigen to bind to its cognate ligand, to inhibit the ability ofCTLA4 to bind to its cognate ligand, to augment T cell responses via theco-stimulatory pathway, to disrupt the ability of B7 to bind to CD28and/or CTLA4, to disrupt the ability of B7 to activate theco-stimulatory pathway, to disrupt the ability of CD80 to bind to CD28and/or CTLA4, to disrupt the ability of CD80 to activate theco-stimulatory pathway, to disrupt the ability of CD86 to bind to CD28and/or CTLA4, to disrupt the ability of CD86 to activate theco-stimulatory pathway, and to disrupt the co-stimulatory pathway, ingeneral from being activated. This necessarily includes small moleculeinhibitors of CD28, CD80, CD86, CTLA4, among other members of theco-stimulatory pathway; antibodies directed to CD28, CD80, CD86, CTLA4,among other members of the co-stimulatory pathway; antisense moleculesdirected against CD28, CD80, CD86, CTLA4, among other members of theco-stimulatory pathway; adnectins directed against CD28, CD80, CD86,CTLA4, among other members of the co-stimulatory pathway, RNAiinhibitors (both single and double stranded) of CD28, CD80, CD86, CTLA4,among other members of the co-stimulatory pathway, among otheranti-CTLA4 antagonists.

Other immune-checkpoint inhibitors include lymphocyte activation gene-3(LAG-3) inhibitors, such as IMP321, a soluble Ig fusion protein(Brignone et al., 2007, J. Immunol. 179:4202-4211). Otherimmune-checkpoint inhibitors include B7 inhibitors, such as B7-H3 andB7-H4 inhibitors. In particular, the anti-B7-H3 antibody MGA271 (Loo etal., 2012, Clin. Cancer Res. July 15 (18) 3834). Also included are TIM3(T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcadeet al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J.Exp. Med. 207:2187-94).

Non-limiting examples of immune checkpoint inhibitors (with checkpointtargets and manufacturers noted in parantheses) can include: MGA271(B7-H3: MacroGenics); ipilimumab (CTLA-4; Bristol Meyers Squibb);pembrolizumab (PD-1; Merck); nivolumab (PD-1; Bristol Meyers Squibb);atezolizumab (PD-L1; Genentech); galiximab (B7.1; Biogen); IMP321 (LAG3:Immuntep); BMS-986016 (LAG3; Bristol Meyers Squibb); SMB-663513 (CD137;Bristol-Meyers Squibb); PF-05082566 (CD137; Pfizer); IPH2101 (KIR;Innate Pharma); KW-0761 (CCR4; Kyowa Kirin); CDX-1127 (CD27; CeilDex);MEDI-6769 (Ox40; Medimmune); CP-870,893 (CD40; Genentech); tremelimumab(CTLA-4; Medimmune); pidilizumab (PD-1; Medivation); MPDL3280A (PD-L1;Roche); MEDI4736 (PD-L1; AstraZeneca); MSB0010718C (PD-L1; EMD Serono);AUNP12 (PD-1; Aurigene); avelumab (PD-L1; Merck); durvalumab (PD-L1;Medimmune).

In some embodiments of any of the aspects, an inhibitor of an immunecheckpoint protein can be an antibody, antibody reagent, antigen-bindingfragment thereof, or CAR that specifically binds to at least one immunecheckpoint protein. Such inhibitors are known in the art. In someembodiments of any of the aspects, inhibitors of an immune checkpointprotein can be inhibitory nucleic acid molecules. In some embodiments ofany of the aspects, an inhibitory nucleic acid molecule can be a miRNA,iRNA, amiRNA, RNAi molecule or the like.

In some embodiments of any of the aspects, an inhibitor of immunecheckpoint proteins can be a natural ligand thereof and/or an inhibitorderived from a natural ligand, e.g., PD-L1 or B7.

In some embodiments of any of the aspects, multiple inhibitors of immunecheckpoint protein(s) can be used in the methods and compositionsdescribed herein, e.g., two or more inhibitors that target the sameimmune checkpoint protein, or at least one inhibitor that targets afirst immune checkpoint protein and at least one inhibitor that targetsat least one additional immune checkpoint protein.

In some embodiments of any of the aspects, the a) inhibitor of CHI3L1,inhibitor of Sema7A, inhibitor of β1-integrin, or agonist of PlexinC1and b) the inhibitor of an immune checkpoint protein are present in thesame bivalent antibody reagent. In some embodiments of any of theaspects, the inhibitor of CHI3L1 and the inhibitor of an immunecheckpoint protein are present in the same bivalent antibody reagent. Insome embodiments of any of the aspects, the immune checkpoint proteintargeted by such a bivalent antibody reagent is PD-1 or CTLA4.

As used herein, the term “bivalent antibody reagent” refers to anantibody reagent that comprises a first antigen-binding domain which hasbinding specificity for a first target, and a second antigen-bindingdomain which has binding specificity for a second target, i.e., theagent has specificity for two targets, e.g., CHI3L1 and PD-1, or CHI3L1and CTLA4. The first target and the second target are not the same(i.e., are different targets (e.g., proteins)). In some embodiments ofany of the aspects, the different targets can be co-expressed on thesame cell. In some embodiments of any of the aspects, a bivalentantibody reagent polypeptide agent can bind targets present on a singlecell (heterophilic binding in cis), and/or bind one target on one celland the other on another cell (heterophilic binding in trans). In someembodiments of any of the aspects, a bivalent antibody reagentpolypeptide agent can bind one or more soluble, extracellular targets.Bivalent antibody reagents comprising antigen-binding portions ofantibodies specific for two different antigens can be readilyconstructed by one of skill in the art. Generally, sequences encodingthe antigen-binding domain of a first antibody characterized and knownto bind a desired epitope on one antigen can be joined, either directly,or through any of a variety of linkers as known to the ordinarilyskilled artisan, to sequences encoding the antigen-binding domain of asecond antibody characterized and known to bind a desired epitope on asecond antigen. Such sequences can be inserted into an appropriatevector and introduced to a cell to produce the bivalent antibodypolypeptide by methods known to those of ordinary skill in the art.

In some embodiments of any of the aspects, any of the antibodies,antigen-binding fragments thereof, or antibody reagents described hereincan be antibody-drug conjugates. In particular embodiments, anantibody-drug conjugate comprises an antibody, antibody reagent, orantigen-binding portion thereof as described herein. The drug can be,e.g., a chemotherapeutic molecule as described elsewhere herein. In someembodiments of any of the aspects, the antibody-drug conjugate comprisesa chemotherapeutic agent directly conjugated and/or bound to an antibodyor antigen-binding portion thereof. In some embodiments of any of theaspects, binding can be non-covalent, e.g., by hydrogen bonding,electrostatic, or van der Waals interactions; however, binding may alsobe covalent. By “conjugated” is meant the covalent linkage of at leasttwo molecules. In some embodiments of any of the aspects, thecomposition can be an antibody-drug conjugate.

In some embodiments of any of the aspects, an antibody, antibodyreagent, or antigen-binding portion thereof can be bound to and/orconjugated to multiple chemotherapeutic molecules. In some embodimentsof any of the aspects, an antibody-drug conjugate can be bound to and/orconjugated to multiple chemotherapeutic molecules. In some embodimentsof any of the aspects, the ratio of a given chemotherapeutic molecule toan antibody or antigen-binding portion thereof can be from about 1:1 toabout 1,000:1, e.g., a single antibody reagent molecule can be linkedto, conjugated to, etc. from about 1 to about 1,000 individualchemotherapeutic molecules.

In some embodiments of any of the aspects, an antibody, orantigen-binding portion thereof, and the chemotherapeutic agent can bepresent in a scaffold material. Scaffold materials suitable for use intherapeutic compositions are known in the art and can include, but arenot limited to, a nanoparticle; a matrix; a hydrogel; and a biomatcrial,biocompatiblc, and/or biodegradable scaffold material. As used herein,the term “nanoparticle” refers to particles that are on the order ofabout 10⁻⁹ or one to several billionths of a meter. The term“nanoparticle” includes nanospheres; nanorods; nanoshells; andnanoprisms; these nanoparticles may be part of a nanonetwork.

The term “nanoparticles” also encompasses liposomes and lipid particleshaving the size of a nanoparticle. As used herein, the term “matrix”refers to a 3-dimensional structure comprising the components of acomposition described herein (e.g., an antibody or antigen-bindingportion thereof). Non-limiting examples of matrix structures includefoams; hydrogels; electrospun fibers; gels; fiber mats; sponges;3-dimensional scaffolds; non-woven mats; woven materials; knitmaterials; fiber bundles; and fibers and other material formats (See,e.g., Rockwood et al. Nature Protocols 2011 6:1612-1631 and US PatentPublications 2011/0167602; 2011/0009960; 2012/0296352; and U.S. Pat. No.8,172,901; each of which is incorporated by reference herein in itsentirety). The structure of the matrix can be selected by one of skillin the art depending upon the intended application of the composition,e.g., electrospun matrices can have greater surface area than foams.

In some embodiments of any of the aspects, the scaffold is a hydrogel.As used herein, the term “hydrogel” refers to a three-dimensionalpolymeric structure that is insoluble in water but which is capable ofabsorbing and retaining large quantities of water to form a stable,often soft and pliable, structure. In some embodiments of any of theaspects, water can penetrate in between the polymer chains of thepolymer network, subsequently causing swelling and the formation of ahydrogel. In general, hydrogels are superabsorbent. Hydrogels have manydesirable properties for biomedical applications. For example, they canbe made nontoxic and compatible with tissue, and they are highlypermeable to water, ions, and small molecules. Hydrogels aresuper-absorbent (they can contain over 99% water) and can be comprisedof natural (e.g., silk) or synthetic polymers, e.g., PEG.

As used herein, “biomaterial” refers to a material that is biocompatibleand biodegradable. As used herein, the term “biocompatible” refers tosubstances that are not toxic to cells. In some embodiments of any ofthe aspects, a substance is considered to be “biocompatible” if itsaddition to cells in vitro results in less than or equal toapproximately 20% cell death. In some embodiments of any of the aspects,a substance is considered to be “biocompatible” if its addition to cellsin vivo does not induce inflammation and/or other adverse effects invivo. As used herein, the term “biodegradable” refers to substances thatare degraded under physiological conditions. In some embodiments of anyof the aspects, a biodegradable substance is a substance that is brokendown by cellular machinery. In some embodiments of any of the aspects, abiodegradable substance is a substance that is broken down by chemicalprocesses.

In one aspect of any of the embodiments, described herein is acomposition comprising a) an inhibitor of an immune checkpoint proteinand b) at least one of an inhibitor of CHI3L1, an inhibitor ofβ1-integrin, and inhibitor of Sema7A, and an agonist of Plexin C1. Inone aspect of any of the embodiments, described herein is thecombination of a composition comprising an inhibitor of an immunecheckpoint protein and a composition comprising b) at least one of aninhibitor of CHI3L1, an inhibitor of β1-integrin, and inhibitor ofScma7A, and an agonist of Plexin C1 (e.g., combined into a singlecomposition or prepared as separate compositions for use or packaging asa paired unit). In some embodiments of any of the aspects, thecomposition can comprise, one, two, three, or all of an inhibitor ofCHI3L1, an inhibitor of β1-integrin, and inhibitor of Sema7A, and anagonist of Plexin C1.

In one aspect of any of the embodiments, described herein is acomposition comprising an inhibitor of CHI3L1 and an inhibitor of animmune checkpoint protein. In one aspect of any of the embodiments,described herein is the combination of a composition comprising aninhibitor of CHI3L1 and a composition comprising an inhibitor of animmune checkpoint protein (e.g., combined into a single composition orprepared as separate compositions for use or packaging as a pairedunit). In some embodiments of any of the aspects, the composition is apharmaceutical composition. As used herein, the term “pharmaceuticalcomposition” refers to the active agent in combination with apharmaceutically acceptable carrier accepted for use in thepharmaceutical industry. The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in the artand need not be limited based on formulation. Typically, suchcompositions are prepared as injectable either as liquid solutions orsuspensions, however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified or presented as a liposome composition. The active ingredientcan be mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient and in amounts suitable for use inthe therapeutic methods described herein. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents and the like which enhance ormaintain the effectiveness of the active ingredient. The therapeuticcomposition as described herein can include pharmaceutically acceptablesalts of the components therein. Pharmaceutically acceptable saltsinclude the acid addition salts (formed with the free amino groups ofthe polypeptide) that are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, tartaric, mandelic and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like. Physiologically tolerablecarriers are well known in the art. Exemplary liquid carriers aresterile aqueous solutions that contain no materials in addition to theactive ingredients and water, or contain a buffer such as sodiumphosphate at physiological pH value, physiological saline or both, suchas phosphate-buffered saline. Still further, aqueous carriers cancontain more than one buffer salt, as well as salts such as sodium andpotassium chlorides, dextrose, polyethylene glycol and other solutes.Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions. The amount of an active agent used in the invention that willbe effective in the treatment of a particular disorder or condition willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques.

In some embodiments of any of the aspects, the composition as describedherein can be a lyophilisate.

In some embodiments of any of the aspects, the technology describedherein relates to a syringe or catheter, including an organ-specificcatheter (e.g., renal catheter, biliary catheter, cardiac catheter,etc.), comprising a therapeutically effective amount of a compositiondescribed herein.

In one aspect, described herein is a kit comprising one or morecompositions as described herein, e.g., a composition comprising aninhibitor of CHI3L1 and a composition comprising an inhibitor of animmune checkpoint protein; or a composition comprising both an inhibitorof CHI3L1 and composition comprising an inhibitor of an immunecheckpoint protein. A kit is any manufacture (e.g., a package orcontainer) comprising at least one reagent, e.g., an antibody, themanufacture being promoted, distributed, or sold as a unit forperforming the methods described herein. In some embodiments of any ofthe aspects, the antibody, antibody reagent, antigen-binding fragmentthereof as described herein is immobilized on a solid support. In someembodiments of any of the aspects, the solid support comprises aparticle, a bead, a polymer, or a substrate. In some embodiments of anyof the aspects, the antibody, antibody reagent or antigen-bindingfragment thereof is detectably labeled.

The kits described herein can optionally comprise additional componentsuseful for performing the methods described herein. By way of example,the kit can comprise fluids (e.g., buffers) suitable for composition theone or more reagents described herein, an instructional material whichdescribes performance of a method as described herein, and the like. Akit can further comprise devices and/or reagents for delivery of thecomposition as described herein. Additionally, the kit may comprise aninstruction leaflet and/or may provide information as to the relevanceof the obtained results.

As used herein, the phrase “therapeutically effective amount”,“effective amount” or “effective dose” refers to an amount that providesa therapeutic or aesthetic benefit in the treatment, prevention, ormanagement of a tumor or malignancy, e.g., an amount that provides astatistically significant decrease in at least one symptom, sign, ormarker of a tumor or malignancy. Determination of a therapeuticallyeffective amount is well within the capability of those skilled in theart. Generally, a therapeutically effective amount can vary with thesubject's history, age, condition, sex, as well as the severity and typeof the medical condition in the subject, and administration of otherpharmaceutically active agents

In one aspect, the technology described herein relates to a methodcomprising administering a combination of agents described herein to asubject. In one aspect, the technology described herein relates to amethod comprising administering an inhibitor of CHI3L1 and an inhibitorof an immune checkpoint protein to a subject. In some embodiments of anyof the aspects, the subject is in need of treatment for a cancer and/ormalignancy. In some embodiments of any of the aspects, the subject is inneed of treatment for: prostate cancer, colon cancer, rectal cancer,ovarian cancer, kidney cancer, breast cancer, glioblastoma, melanoma,malignant melanoma, and lung cancer. In some embodiments of any of theaspects, the method is a method of treating a subject. In someembodiments of any of the aspects, the method is a method of treating acancer in a subject.

As used herein, the term “cancer” relates generally to a class ofdiseases or conditions in which abnormal cells divide without controland can invade nearby tissues. Cancer cells can also spread to otherparts of the body through the blood and lymph systems. There are severalmain types of cancer. Carcinoma is a cancer that begins in the skin orin tissues that line or cover internal organs. Sarcoma is a cancer thatbegins in bone, cartilage, fat, muscle, blood vessels, or otherconnective or supportive tissue. Leukemia is a cancer that starts inblood-forming tissue such as the bone marrow, and causes large numbersof abnormal blood cells to be produced and enter the blood. Lymphoma andmultiple myeloma are cancers that begin in the cells of the immunesystem. Central nervous system cancers are cancers that begin in thetissues of the brain and spinal cord.

In some embodiments of any of the aspects, the cancer is a primarycancer. In some embodiments of any of the aspects, the cancer is amalignant cancer. As used herein, the term “malignant” refers to acancer in which a group of tumor cells display one or more ofuncontrolled growth (i.e., division beyond normal limits), invasion(i.e., intrusion on and destruction of adjacent tissues), and metastasis(i.e., spread to other locations in the body via lymph or blood). Asused herein, the term “metastasize” refers to the spread of cancer fromone part of the body to another. A tumor formed by cells that havespread is called a “metastatic tumor” or a “metastasis.” The metastatictumor contains cells that are like those in the original (primary)tumor.

As used herein, the term “benign” or “non-malignant” refers to tumorsthat may grow larger but do not spread to other parts of the body.Benign tumors are self-limited and typically do not invade ormetastasize.

A “cancer cell” or “tumor cell” refers to an individual cell of acancerous growth or tissue. A tumor refers generally to a swelling orlesion formed by an abnormal growth of cells, which may be benign,pre-malignant, or malignant. Most cancer cells form tumors, but some,e.g., leukemia, do not necessarily form tumors. For those cancer cellsthat form tumors, the terms cancer (cell) and tumor (cell) are usedinterchangeably.

A subject that has a cancer or a tumor is a subject having objectivelymeasurable cancer cells present in the subject's body. Included in thisdefinition are malignant, actively proliferative cancers, as well aspotentially dormant tumors or micrometastatses. Cancers which migratefrom their original location and seed other vital organs can eventuallylead to the death of the subject through the functional deterioration ofthe affected organs. Hemopoietic cancers, such as leukemia, are able toout-compete the normal hemopoietic compartments in a subject, therebyleading to hemopoietic failure (in the form of anemia, thrombocytopeniaand neutropenia) ultimately causing death.

Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer;bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancerof the peritoneum; cervical cancer; choriocarcinoma; colon and rectumcancer; connective tissue cancer; cancer of the digestive system;endometrial cancer; esophageal cancer; eye cancer; cancer of the headand neck; gastric cancer (including gastrointestinal cancer);glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelialneoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer;lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung);lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma;myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth,and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; salivary gland carcinoma; sarcoma; skin cancer;squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer;uterine or endometrial cancer; cancer of the urinary system; vulvalcancer; as well as other carcinomas and sarcomas; as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.

A “cancer cell” is a cancerous, pre-cancerous, or transformed cell,either in vivo, ex vivo, or in tissue culture, that has spontaneous orinduced phenotypic changes that do not necessarily involve the uptake ofnew genetic material. Although transformation can arise from infectionwith a transforming virus and incorporation of new genomic nucleic acid,or uptake of exogenous nucleic acid, it can also arise spontaneously orfollowing exposure to a carcinogen, thereby mutating an endogenous gene.Transformation/cancer is associated with, e.g., morphological changes,immortalization of cells, aberrant growth control, foci formation,anchorage independence, malignancy, loss of contact inhibition anddensity limitation of growth, growth factor or serum independence, tumorspecific markers, invasiveness or metastasis, and tumor growth insuitable animal hosts such as nude mice.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.Patients or subjects include any subset of the foregoing, e.g., all ofthe above, but excluding one or more groups or species such as humans,primates or rodents. In certain embodiments, the subject is a mammal,e.g., a primate, e.g., a human. The terms, “patient”, “individual” and“subject” are used interchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used, for example, as subjects that represent animalmodels of, for example, various cancers. In addition, the methodsdescribed herein can be used to treat domesticated animals and/or pets.A subject can be male or female.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatment(e.g., a cancer) or one or more complications related to such acondition, and optionally, but need not have already undergone treatmentfor a condition or the one or more complications related to thecondition. Alternatively, a subject can also be one who has not beenpreviously diagnosed as having a condition in need of treatment or oneor more complications related to such a condition. For example, asubject can be one who exhibits one or more risk factors for a conditionor one or more complications related to a condition or a subject whodoes not exhibit risk factors. A “subject in need” of treatment for aparticular condition can be a subject having that condition, diagnosedas having that condition, or at risk of developing that condition.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” when used in reference to a disease, disorder or medicalcondition, refer to therapeutic treatments for a condition, wherein theobject is to reverse, alleviate, ameliorate, inhibit, slow down or stopthe progression or severity of a symptom or condition. The term“treating” includes reducing or alleviating at least one adverse effector symptom of a condition. Treatment is generally “effective” if one ormore symptoms or clinical markers are reduced. Alternatively, treatmentis “effective” if the progression of a condition is reduced or halted.That is, “treatment” includes not just the improvement of symptoms ormarkers, but also a cessation or at least slowing of progress orworsening of symptoms that would be expected in the absence oftreatment. Beneficial or desired clinical results include, but are notlimited to, alleviation of one or more symptom(s), diminishment ofextent of the deficit, stabilized (i.e., not worsening) state of a tumoror malignancy, delay or slowing of tumor growth and/or metastasis, andan increased lifespan as compared to that expected in the absence oftreatment. As used herein, the term “administering,” refers to theplacement of an agent, including but not limited to, an antibody,antibody reagent, antigen-binding portion thereof, or CAR as describedherein or a nucleic acid encoding an antibody, antibody reagent,antigen-binding portion thereof, or CAR, or a cell comprising such anagent, as described herein into a subject by a method or route whichresults in at least partial localization of the agents at a desiredsite. The pharmaceutical composition comprising an antibody, antibodyreagent, antigen-binding portion thereof, or CAR as described herein ora nucleic acid encoding an antibody, antibody reagent, antigen-bindingportion thereof, or CAR, or a cell comprising such an agent as describedherein disclosed herein can be administered by any appropriate routewhich results in an effective treatment in the subject.

In some embodiments of any of the aspects, the methods described hereinrelate to CAR-T cell therapy. CAR-T cell and related therapies relate toadoptive cell transfer of immune cells (e.g., T cells) expressing a CARthat binds specifically to a targeted cell type (e.g., cancer cells) totreat a subject. In some embodiments of any of the aspects, the cellsadministered as part of the therapy can be autologous to the subject. Insome embodiments of any of the aspects, the cells administered as partof the therapy are not autologous to the subject. In some embodiments ofany of the aspects, the cells are engineered and/or genetically modifiedto express the CAR. Further discussion of CAR-T therapies can be found,e.g., in Maus et al. Blood 2014 123:2624-35; Reardon et al.Neuro-Oncology 2014 16:1441-1458; Hoyos et al. Haematologica 201297:1622; Byrd et al. J Clin Oncol 2014 32:3039-47; Maher et al. CancerRes 2009 69:4559-4562; and Tamada et al. Clin Cancer Res 201218:6436-6445; each of which is incorporated by reference herein in itsentirety.

The administration of the compositions contemplated herein may becarried out in any convenient manner, including by aerosol inhalation,injection, ingestion, transfusion, implantation or transplantation. In apreferred embodiment, compositions are administered parenterally. Thephrases “parenteral administration” and “administered parenterally” asused herein refers to modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravascular, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intratumoral, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion. In one embodiment, the compositions contemplatedherein are administered to a subject by direct injection into a tumor,lymph node, or site of infection.

It can generally be stated that a pharmaceutical composition comprisingthe cells, e.g., T cells or immune cells, described herein may beadministered at a dosage of 10² to 10¹⁰ cells/kg body weight, preferably10⁵ to 10⁶ cells/kg body weight, including all integer values withinthose ranges. The number of cells will depend upon the ultimate use forwhich the composition is intended as will the type of cells includedtherein. For uses provided herein, the cells are generally in a volumeof a liter or less, can be 500 mLs or less, even 250 mLs or 100 mLs orless. Hence the density of the desired cells is typically greater than10⁶ cells/ml and generally is greater than 10⁷ cells/ml, generally 10⁸cells/ml or greater. The clinically relevant number of immune cells canbe apportioned into multiple infusions that cumulatively equal or exceed10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, or 10¹² cells. In some aspects ofthe present invention, particularly since all the infused cells will beredirected to a particular target antigen, lower numbers of cells, inthe range of 10⁶/kilogram (10⁶-10¹¹ per patient) may be administered.CAR expressing cell compositions may be administered multiple times atdosages within these ranges. The cells may be allogeneic, syngeneic,xenogeneic, or autologous to the patient undergoing therapy. If desired,the treatment may also include administration of mitogens (e.g., PHA) orlymphokines, cytokines, and/or chemokines (e.g., IFN-γ, IL-2, IL-12,TNF-alpha, IL-18, and TNF-beta, GM-CSF, IL-4, IL-13, Flt3-L, RANTES,MIP1α, etc.) as described herein to enhance induction of the immuneresponse. In some embodiments of any of the aspects, the dosage can befrom about 1×10⁵ cells to about 1×10⁸ cells per kg of body weight. Insome embodiments of any of the aspects, the dosage can be from about1×10⁶ cells to about 1×10⁷ cells per kg of body weight. In someembodiments of any of the aspects, the dosage can be about 1×10⁶ cellsper kg of body weight. In some embodiments of any of the aspects, onedose of cells can be administered. In some embodiments of any of theaspects, the dose of cells can be repeated, e.g., once, twice, or more.In some embodiments of any of the aspects, the dose of cells can beadministered on, e.g., a daily, weekly, or monthly basis.

The dosage ranges for the agent depend upon the potency, and encompassamounts large enough to produce the desired effect e.g., slowing oftumor growth or a reduction in tumor size. The dosage should not be solarge as to cause unacceptable adverse side effects. Generally, thedosage will vary with the age, condition, and sex of the patient and canbe determined by one of skill in the art. The dosage can also beadjusted by the individual physician in the event of any complication.In some embodiments of any of the aspects, the dosage ranges from 0.001mg/kg body weight to 0.5 mg/kg body weight. In some embodiments of anyof the aspects, the dose range is from 5 μg/kg body weight to 100 μg/kgbody weight. Alternatively, the dose range can be titrated to maintainserum levels between 1 μg/mL and 1000 μg/mL. For systemicadministration, subjects can be administered a therapeutic amount, suchas, e.g., 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50mg/kg, or more.

Administration of the doses recited above can be repeated. In someembodiments of any of the aspects, the doses are given once a day, ormultiple times a day, for example but not limited to three times a day.In some embodiments, the doses recited above are administered daily forseveral weeks or months. The duration of treatment depends upon thesubject's clinical progress and responsiveness to therapy.

In some embodiments of any of the aspects, the dose can be from about 2mg/kg to about 15 mg/kg. In some embodiments of any of the aspects, thedose can be about 2 mg/kg. In some embodiments of any of the aspects,the dose can be about 4 mg/kg. In some embodiments of any of theaspects, the dose can be about 5 mg/kg. In some embodiments of any ofthe aspects, the dose can be about 6 mg/kg. In some embodiments of anyof the aspects, the dose can be about 8 mg/kg. In some embodiments ofany of the aspects, the dose can be about 10 mg/kg. In some embodimentsof any of the aspects, the dose can be about 15 mg/kg. In someembodiments of any of the aspects, the dose can be from about 100 mg/m²to about 700 mg/m². In some embodiments of any of the aspects, the dosecan be about 250 mg/m². In some embodiments of any of the aspects, thedose can be about 375 mg/m². In some embodiments of any of the aspects,the dose can be about 400 mg/m². In some embodiments of any of theaspects, the dose can be about 500 mg/m².

In some embodiments of any of the aspects, the dose can be administeredintravenously. In some embodiments of any of the aspects, theintravenous administration can be an infusion occurring over a period offrom about 10 minute to about 3 hours. In some embodiments of any of theaspects, the intravenous administration can be an infusion occurringover a period of from about 30 minutes to about 90 minutes.

In some embodiments the dose can be administered about weekly. In someembodiments of any of the aspects, the dose can be administered weekly.In some embodiments of any of the aspects, the dose can be administeredweekly for from about 12 weeks to about 18 weeks. In some embodimentsthe dose can be administered about every 2 weeks. In some embodimentsthe dose can be administered about every 3 weeks. In some embodiments ofany of the aspects, the dose can be from about 2 mg/kg to about 15 mg/kgadministered about every 2 weeks. In some embodiments of any of theaspects, the dose can be from about 2 mg/kg to about 15 mg/kgadministered about every 3 weeks. In some embodiments of any of theaspects, the dose can be from about 2 mg/kg to about 15 mg/kgadministered intravenously about every 2 weeks. In some embodiments ofany of the aspects, the dose can be from about 2 mg/kg to about 15 mg/kgadministered intravenously about every 3 weeks. In some embodiments ofany of the aspects, the dose can be from about 200 mg/m² to about 400mg/m² administered intravenously about every week. In some embodimentsof any of the aspects, the dose can be from about 200 mg/m² to about 400mg/m² administered intravenously about every 2 weeks. In someembodiments of any of the aspects, the dose can be from about 200 mg/m²to about 400 mg/m² administered intravenously about every 3 weeks. Insome embodiments of any of the aspects, a total of from about 2 to about10 doses are administered. In some embodiments of any of the aspects, atotal of 4 doses are administered. In some embodiments of any of theaspects, a total of 5 doses are administered. In some embodiments of anyof the aspects, a total of 6 doses are administered. In some embodimentsof any of the aspects, a total of 7 doses are administered. In someembodiments of any of the aspects, a total of 8 doses are administered.In some embodiments of any of the aspects, the administration occurs fora total of from about 4 weeks to about 12 weeks. In some embodiments ofany of the aspects, the administration occurs for a total of about 6weeks. In some embodiments of any of the aspects, the administrationoccurs for a total of about 8 weeks. In some embodiments of any of theaspects, the administration occurs for a total of about 12 weeks. Insome embodiments of any of the aspects, the initial dose can be fromabout 1.5 to about 2.5 fold greater than subsequent doses.

In some embodiments of any of the aspects, the dose can be from about 1mg to about 2000 mg. In some embodiments of any of the aspects, the dosecan be about 3 mg. In some embodiments of any of the aspects, the dosecan be about 10 mg. In some embodiments of any of the aspects, the dosecan be about 30 mg. In some embodiments of any of the aspects, the dosecan be about 1000 mg. In some embodiments of any of the aspects, thedose can be about 2000 mg. In some embodiments of any of the aspects,the dose can be about 3 mg given by intravenous infusion daily. In someembodiments of any of the aspects, the dose can be about 10 mg given byintravenous infusion daily. In some embodiments of any of the aspects,the dose can be about 30 mg given by intravenous infusion three timesper week.

A therapeutically effective amount is an amount of an agent that issufficient to produce a statistically significant, measurable change intumor size, tumor growth etc. (efficacy measurements are described belowherein). Such effective amounts can be gauged in clinical trials as wellas animal studies.

An agent can be administered intravenously by injection or by gradualinfusion over time. Given an appropriate formulation for a given route,for example, agents useful in the methods and compositions describedherein can be administered intravenously, intranasally, by inhalation,intraperitoneally, intramuscularly, subcutaneously, intracavity, and canbe delivered by peristaltic means, if desired, or by other means knownby those skilled in the art. It is preferred that the compounds usedherein are administered orally, intravenously or intramuscularly to apatient having cancer. Local administration directly to a tumor mass isalso specifically contemplated.

Therapeutic compositions containing at least one agent can beconventionally administered in a unit dose, for example. The term “unitdose” when used in reference to a therapeutic composition refers tophysically discrete units suitable as unitary dosage for the subject,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required physiologically acceptable diluent, i.e., carrier, orvehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered and timing depends on the subject to be treated,capacity of the subject's system to utilize the active ingredient, anddegree of therapeutic effect desired.

Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner and are particular to eachindividual. However, suitable dosage ranges for systemic application aredisclosed herein and depend on the route of administration. Suitableregimes for administration are also variable, but are typified by aninitial administration followed by repeated doses at one or more hourintervals by a subsequent injection or other administration.Alternatively, continuous intravenous infusion sufficient to maintainconcentrations in the blood in the ranges specified for in vivotherapies are contemplated.

The multiple agents of the combinations described herein can beadministered concurrently or sequentially. The multiple agents of thecombinations described herein can be administered in the same orseparate formulations. The multiple agents of the combinations describedherein can be administered by the same or different routes ofadministration. In some embodiments of any of the aspects, multipleagents of the combinations described herein can be administered on thesame day, within 2 days of each other, within 3 days of each other,within 4 days of each other, within 5 days of each other, within 6 daysof each other, or within 7 days of each other. In some embodiments ofany of the aspects, for any pair-wise combination of agents, thesecond-administered agent is administered during the period in which thefirst-administered agent or its consequences are still detectable in thesubject.

The inhibitor of CHI3L1 and the inhibitor of an immune checkpointprotein can be administered concurrently or sequentially. The inhibitorof CHI3L1 and the inhibitor of an immune checkpoint protein can beadministered in the same or separate formulations. The inhibitor ofCHI3L1 and the inhibitor of an immune checkpoint protein can beadministered by the same or different routes of administration. In someembodiments of any of the aspects, the inhibitor of CHI3L1 and theinhibitor of an immune checkpoint protein can be administered on thesame day, within 2 days of each other, within 3 days of each other,within 4 days of each other, within 5 days of each other, within 6 daysof each other, or within 7 days of each other. In some embodiments ofany of the aspects the second-administered agent is administered duringthe period in which the first-administered agent is still detectable inthe subject.

In some embodiments of any of the aspects, the methods further compriseadministering the pharmaceutical composition(s) described herein alongwith one or more additional chemotherapeutic agents, biologics, drugs,or treatments as part of a combinatorial therapy. In some suchembodiments, the chemotherapeutic agent biologic, drug, or treatment isselected from the group consisting of: radiation therapy, surgery,antibody reagents, and/or small molecules.

In some embodiments of the methods described herein, the methods furthercomprise administering one or more chemotherapeutic agents to thesubject being administered the pharmaceutical composition describedherein. Non-limiting examples of chemotherapeutic agents can includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including the synthetic analoguetopotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegall; dynemicin,including dynemicin A; bisphosphonates, such as clodronate; anesperamicin; as well as ncocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (Tykerb®); inhibitors of PKC-alpha, Raf,H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any one of the above.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins, such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof.

As used herein, the terms “chemotherapy” or “chemotherapeutic agent”refer to any chemical agent with therapeutic usefulness in the treatmentof diseases characterized by abnormal cell growth. Such diseases includetumors, neoplasms and cancer as well as diseases characterized byhyperplastic growth. Chemotherapeutic agents as used herein encompassboth chemical and biological agents. These agents function to inhibit acellular activity upon which the cancer cell depends for continuedsurvival. Categories of chemotherapeutic agents includealkylating/alkaloid agents, antimetabolites, hormones or hormoneanalogs, and miscellaneous antineoplastic drugs. Most if not all ofthese agents are directly toxic to cancer cells and do not requireimmune stimulation. In one embodiment, a chemotherapeutic agent is anagent of use in treating neoplasms such as solid tumors. In oneembodiment, a chemotherapeutic agent is a radioactive molecule. One ofskill in the art can readily identify a chemotherapeutic agent of use(e.g., see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 inHarrison's Principles of Internal Medicine, 14th edition; Perry et al.,Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^(nd) Edition, 2000Churchill Livingstone, Inc; Baltzer L, Berkery R (eds)). The bispecificand multispecific polypeptide agents described herein can be used inconjunction with additional chemotherapeutic agents.

By “radiation therapy” is meant the use of directed gamma rays or betarays to induce sufficient damage to a cell so as to limit its ability tofunction normally or to destroy the cell altogether. It will beappreciated that there will be many ways known in the art to determinethe dosage and duration of treatment. Typical treatments are given as aone time administration and typical dosages range from 10 to 200 units(Grays) per day.

In some embodiments of any of the aspects, the methods described hereincan further comprise administering an additional immunotherapy to thesubject. As used herein, “immunotherapy” refers to a diverse set oftherapeutic strategies designed to induce the patient's own immunesystem to fight the tumor, and include, but are not limited to,intravesical BCG immunotherapy for superficial bladder cancer, vaccinesto generate specific immune responses, such as for malignant melanomaand renal cell carcinoma, the use of Sipuleucel-T for prostate cancer,in which dendritic cells from the patient are loaded with prostatic acidphosphatase peptides to induce a specific immune response againstprostate-derived cells, administration of cytokines, growth factorsand/or signaling molecules that stimulate one or more immune cell type(e.g., interleukins), ex vivo expansion and/or stimulation oflymphocytes and/or dendritic cell specific for a tumor antigen prior toreintroduction to the patient, imiquimod, adoptive cell transfer, and/orthe methods described, e.g., in International Patent Publication WO2003/063792 and U.S. Pat. No. 8,329,660. In some embodiments of any ofthe aspects, the immunotherapy stimulates NK responses. In someembodiments of any of the aspects, the immunotherapy is an adoptive celltransfer approach, i.e., adoptive immunotherapy.

In some embodiments of any of the aspects, the methods described hereincan further comprise administering an additional antibody, antibodyreagent, antigen-binding portion thereof, or T cell comprising a CAR tothe subject. In some embodiments of any of the aspects, the methodsdescribed herein can further comprise administering cytokine to thesubject. Antibody- and cytokine-based therapies are known in the art andcan include, by way of non-limiting example, alemtuzumab; bevacizumab;brentuximab vedotin; cetuximab; gemtuzumab; ibritumomab tiuxetan;ipilimumab; ofatumumab; pantibumumab; rituximab; tositumomab;trastuzumab; interleukin-2, and interferon-alpha.

The efficacy of a given treatment for, e.g., cancer, can be determinedby the skilled clinician. However, a treatment is considered “effectivetreatment,” as the term is used herein, if any one or all of the signsor symptoms of e.g., a tumor are altered in a beneficial manner or otherclinically accepted symptoms are improved, or even ameliorated, e.g., byat least 10% following treatment with an agent as described herein.Efficacy can also be measured by a failure of an individual to worsen asassessed by hospitalization or need for medical interventions (i.e.,progression of the disease is halted). Methods of measuring theseindicators are known to those of skill in the art and/or describedherein.

An effective amount for the treatment of a disease means that amountwhich, when administered to a mammal in need thereof, is sufficient toresult in effective treatment as that term is defined herein, for thatdisease. Efficacy of an agent can be determined by assessing physicalindicators of, for example cancer, e.g., tumor size, tumor mass, tumordensity, angiogenesis, tumor growth rate, etc.

In some embodiments of any of the aspects described herein, a subjectadministered a composition described herein can be a subject determinedto have an elevated level of CHI3L1 or a level of CHI3L1 that isincreased compared to a prior assessment of the level in that subject.In some embodiments of any of the aspects, the elevated level of CHI3L1is the level of circulating CHI3L1. In some embodiments of any of theaspects described herein, a subject administered a composition describedherein can be a subject determined to have cancer cells which areCHI3L1+.

In some embodiments of any of the aspects described herein, the methodcomprising administering a composition as described herein can furthercomprise a first step of identifying a subject having an elevated levelof CHI3L1. In some embodiments of any of the aspects, the elevated levelof CHI3L1 is the level of circulating CHI3L1. In some embodiments of anyof the aspects described herein, the method comprising administering acomposition as described herein can further comprise a first step ofidentifying a subject having cancer cells which are CHI3L1+.

As used herein, a “CHI3L1+” cell is a cell expressing an increased levelof CHI3L1+, e.g., as compared to a healthy cell of the same type or anaverage level of CHI3L1 found in healthy cells of the same type. In someembodiments of any of the aspects, an increased level of CHI3L1 can be alevel which is at least 1.5× the level found in a reference, e.g., 1.5×,2×, 3×, 4×, 5× or greater than the reference level.

In some embodiments of any of the aspects, the expression level ofCHI3L1 can be measured by determining the level of an expression productof the CHI3L1 gene, e.g., a CHI3L1 RNA transcript or a CHI3L1polypeptide. Such molecules can be isolated, derived, or amplified froma biological sample, such as a biofluid. In some embodiments of any ofthe aspects, a detectable signal is generated by the antibody orantigen-binding portion thereof when a CHI3L1 molecule is present. Insome embodiments of any of the aspects, the antibody or antigen-bindingportion thereof is detectably labeled or capable of generating adetectable signal. In some embodiments of any of the aspects, the levelof the CHI3L1 is determined using a method selected from the groupconsisting of: Western blot; immunoprecipitation; enzyme-linkedimmunosorbent assay (ELISA); radioimmunological assay (RIA); sandwichassay; fluorescence in situ hybridization (FISH); immunohistologicalstaining; radioimmunometric assay; immunofluoresence assay; massspectroscopy; FACS; and immunoelectrophoresis assay. In some embodimentsof any of the aspects, the antibody or antigen-binding portion thereofis detectably labeled or generates a detectable signal. In someembodiments of any of the aspects, the expression level of CHI3L1 isnormalized relative to the expression level of one or more referencegenes or reference proteins. In some embodiments of any of the aspects,the reference level of CHI3L1 is the expression level of CHI3L1 in aprior sample obtained from the subject.

In some embodiments of any of the aspects, the level of CHI3L1 can bethe level of CHI3L1 polypeptide. Detection of CHI3L1 polypeptides can beaccording to any method known in the art. Immunological methods todetect CHI3L1 polypeptides in accordance with the present technologyinclude, but are not limited to antibody techniques such asimmunohistochemistry, immunocytochemistry, flow cytometry,fluorescence-activated cell sorting (FACS), immunoblotting,radioimmunoassays, western blotting, immunoprecipitation, enzyme-linkedimmunosorbant assays (ELISA), and derivative techniques that make use ofantibody reagents as described herein.

Immunochemical methods require the use of an antibody reagent specificfor the target molecule (e.g., the antigen or in the embodimentsdescribed herein, a CHI3L1 polypeptide. In some embodiments of any ofthe aspects, the assays, methods, and/or systems described herein cancomprise: an anti-CHI3L1 antibody reagent. In some embodiments of any ofthe aspects, the antibody reagent can be detectably labeled. In someembodiments of any of the aspects, the antibody reagent can be attachedto a solid support (e.g., bound to a solid support). In some embodimentsof any of the aspects, the solid support can comprise a particle(including, but not limited to an agarose or latex bead or particle or amagnetic particle), a bead, a nanoparticle, a polymer, a substrate, aslide, a coverslip, a plate, a dish, a well, a membrane, and/or agrating. The solid support can include many different materialsincluding, but not limited to, polymers, plastics, resins,polysaccharides, silicon or silica based materials, carbon, metals,inorganic glasses, and membranes.

In one embodiment, an assay, method, and/or system as described hereincan comprise an ELISA. In an exemplary embodiment, a first antibodyreagent can be immobilized on a solid support (usually a polystyrenemicro titer plate). The solid support can be contacted with a sampleobtained from a subject, and the antibody reagent will bind (“capture”)antigens for which it is specific (e.g., CHI3L1). The solid support canthen be contacted with a second labeled antibody reagent (e.g., adetection antibody reagent). The detection antibody reagent can, e.g.,comprise a detectable signal, be covalently linked to an enzyme, or canitself be detected by a secondary antibody which is linked to an enzymethrough bio-conjugation. The presence of a signal indicates that boththe first antibody reagent immobilized on the support and the second“detection” antibody reagent have bound to an antigen, i.e., thepresence of a signal indicated the presence of a CHI3L1 molecule.Between each step the plate is typically washed with a mild detergentsolution to remove any proteins or antibodies that are not specificallybound. After the final wash step the plate is developed by adding anenzymatic substrate to produce a visible signal, which indicates thequantity of CHI3L1 polypeptides in the sample. Older ELISAs utilizechromogenic substrates, though newer assays employ fluorogenicsubstrates with much higher sensitivity. There are other different formsof ELISA, which are well known to those skilled in the art.

In one embodiment, the assays, systems, and methods described herein cancomprise a lateral flow immunoassay test (LFIA), also known as theimmunochromatographic assay, or strip test to measure or determine thelevel of CHI3L1 polypeptide in a sample. LFIAs are a simple deviceintended to detect the presence (or absence) of CHI3L1 in a sample.There are currently many LFIA tests used for medical diagnostics eitherfor home testing, point of care testing, or laboratory use. LFIA testsare a form of immunoassay in which the test sample flows along a solidsubstrate via capillary action. After the sample is applied to the teststrip it encounters a colored antibody reagent which mixes with thesample, and if bound to a portion of the sample, transits the substrateencountering lines or zones which have been pretreated with a secondantibody reagent. Depending upon the level of CHI3L1 present in thesample the colored antibody reagent can become bound at the test line orzone. LFIAs are essentially immunoassays adapted to operate along asingle axis to suit the test strip format or a dipstick format. Striptests are extremely versatile and can be easily modified by one skilledin the art for detecting an enormous range of antigens from fluidsamples such as urine, blood, water samples etc. Strip tests are alsoknown as dip stick test, the name bearing from the literal action of“dipping” the test strip into a fluid sample to be tested. LFIA striptest are easy to use, require minimum training and can easily beincluded as components of point-of-care test (POCT) diagnostics to beused on site in the field. LFIA tests can be operated as eithercompetitive or sandwich assays. Sandwich LFIAs are similar to sandwichELISA. The sample first encounters colored particles which are labeledwith antibody reagents specific for a target (e.g., a CHI3L1-specificantibody reagent). The test line will also contain antibody reagents(e.g., a CHI3L1-specific antibody reagent). The test line will show as acolored band in positive samples. In some embodiments of any of theaspects, the lateral flow immunoassay can be a double antibody sandwichassay, a competitive assay, a quantitative assay or variations thereof.There are a number of variations on lateral flow technology. It is alsopossible to apply multiple capture zones to create a multiplex test.

A typical test strip consists of the following components: (1) sampleapplication area comprising an absorbent pad (i. e. the matrix ormaterial) onto which the test sample is applied; (2) conjugate orreagent pad-this contains antibody reagent(s) specific to the targetwhich can be conjugated to colored particles (usually colloidal goldparticles, or latex microspheres); (3) test results area comprising areaction membrane—typically a hydrophobic nitrocellulose or celluloseacetate membrane onto which antibody reagents are immobilized in a lineacross the membrane as a capture zone or test line (a control zone mayalso be present, containing antibodies specific for the antibodyreagents conjugated to the particles or microspheres); and (4) optionalwick or waste reservoir—a further absorbent pad designed to draw thesample across the reaction membrane by capillary action and collect it.The components of the strip are usually fixed to an inert backingmaterial and may be presented in a simple dipstick format or within aplastic casing with a sample port and reaction window showing thecapture and control zones. While not strictly necessary, most tests willincorporate a second line which contains an antibody that picks up freelatex/gold in order to confirm the test has operated correctly.

The use of “dip sticks” or LFIA test strips and other solid supports hasbeen described in the art in the context of an immunoassay for a numberof antigen biomarkers. U.S. Pat. Nos. 4,943,522; 6,485,982; 6,187,598;5,770,460; 5,622,871; 6,565,808, U.S. patent application Ser. No.10/278,676; U.S. Ser. No. 09/579,673 and U.S. Ser. No. 10/717,082, whichare incorporated herein by reference in their entirety, are non-limitingexamples of such lateral flow test devices. Three U.S. patents (U.S.Pat. No. 4,444,880, issued to H. Tom; U.S. Pat. No. 4,305,924, issued toR. N. Piasio; and U.S. Pat. No. 4,135,884, issued to J. T. Shen)describe the use of “dip stick” technology to detect soluble antigensvia immunochemical assays. The apparatuses and methods of these threepatents broadly describe a first component fixed to a solid surface on a“dip stick” which is exposed to a solution containing a soluble antigenthat binds to the component fixed upon the “dip stick,” prior todetection of the component-antigen complex upon the stick. It is withinthe skill of one in the art to modify the teaching of these “dip stick”technologies as necessary for the detection of CHI3L1 polypeptides. Insome embodiments of any of the aspects, the dip stick (or LFIA) can besuitable for use with urine samples. In some embodiments of any of theaspects, a dip stick can be suitable for use with blood samples.

Immunochemistry is a family of techniques based on the use of a specificantibody, wherein antibodies are used to specifically target moleculesinside or on the surface of cells. In some embodiments of any of theaspects, immunohistochemistry (“IHC”) and immunocytochemistry (“ICC”)techniques can be used to detect or measure the levels of CHI3L1polypeptide. IHC is the application of immunochemistry to tissuesections, whereas ICC is the application of immunochemistry to cells ortissue imprints after they have undergone specific cytologicalpreparations such as, for example, liquid-based preparations. In someinstances, signal amplification may be integrated into the particularprotocol, wherein a secondary antibody, that includes a label, followsthe application of an antibody reagent specific for platelets orleukocytes. Typically, for immunohistochemistry, tissue obtained from asubject and fixed by a suitable fixing agent such as alcohol, acetone,and paraformaldehyde, is sectioned and reacted with an antibody.Conventional methods for immunohistochemistry are described in Buchwalowand Bocker (Eds) “Immunohistochemistry: Basics and Methods” Springer(2010): Lin and Prichard “Handbook of Practical Immunohistochemistry”Springer (2011); which are incorporated by reference herein in theirentireties. In some embodiments of any of the aspects,immunocytochemistry may be utilized where, in general, tissue or cellsobtained from a subject are fixed by a suitable fixing agent such asalcohol, acetone, and paraformaldehyde, to which is reacted an antibody.Methods of immunocytological staining of human samples is known to thoseof skill in the art and described, for example, in Burry“Immunocytochemistry: A Practical Guide for Biomedical Research”Springer (2009); which is incorporated by reference herein in itsentirety.

In some embodiments of any of the aspects, one or more of the antibodyreagents described herein can comprise a detectable label and/orcomprise the ability to generate a detectable signal (e.g., bycatalyzing a reaction converting a compound to a detectable product).Detectable labels can comprise, for example, a light-absorbing dye, afluorescent dye, or a radioactive label. Detectable labels, methods ofdetecting them, and methods of incorporating them into an antibodyreagent are well known in the art.

In some embodiments of any of the aspects, detectable labels can includelabels that can be detected by spectroscopic, photochemical,biochemical, immunochemical, electromagnetic, radiochemical, or chemicalmeans, such as fluorescence, chemifluoresence, or chemiluminescence, orany other appropriate means. The detectable labels used in the methodsdescribed herein can be primary labels (where the label comprises amoiety that is directly detectable or that produces a directlydetectable moiety) or secondary labels (where the detectable label bindsto another moiety to produce a detectable signal, e.g., as is common inimmunological labeling using secondary and tertiary antibodies). Thedetectable label can be linked by covalent or non-covalent means to theantibody reagent. Alternatively, a detectable label can be linked suchas by directly labeling a molecule that achieves binding to the antibodyreagent via a ligand-receptor binding pair arrangement or other suchspecific recognition molecules. Detectable labels can include, but arenot limited to radioisotopes, bioluminescent compounds, chromophores,antibodies, chemiluminescent compounds, fluorescent compounds, metalchelates, and enzymes.

In other embodiments, the detection antibody is labeled with afluorescent compound. When the fluorescently labeled antibody is exposedto light of the proper wavelength, its presence can then be detected dueto fluorescence. In some embodiments of any of the aspects, a detectablelabel can be a fluorescent dye molecule, or fluorophore including, butnot limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde,fluorescamine, Cy3™, Cy5™, allophycocyanine, Texas Red, perideninchlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5™,green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC)and Oregon Green™, rhodamine and derivatives (e.g., Texas red andtetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA,CyDyes™, 6-carboxyfhiorescein (commonly known by the abbreviations FAMand F), 6-carboxy-2′,4′,7′,4,7-hexachlorofiuorescein (HEX),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfiuorescein (JOE or J),N,N,N′,N′-tetramethyl-6carboxyrhodamine (TAMRA or T),6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5),6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes,e.g., Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimidedyes, e.g., Hoechst 33258; phenanthridine dyes, e.g., Texas Red;ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes;porphyrin dyes; polymethine dyes, e.g., cyanine dyes such as Cy3, Cy5,etc; BODIPY dyes and quinoline dyes.

In some embodiments of any of the aspects, a detectable label can be aradiolabel including, but not limited to ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, and³³P.

In some embodiments of any of the aspects, a detectable label can be anenzyme including, but not limited to horseradish peroxidase and alkalinephosphatase. An enzymatic label can produce, for example, achemiluminescent signal, a color signal, or a fluorescent signal.Enzymes contemplated for use to detectably label an antibody reagentinclude, but are not limited to, malate dehydrogenase, staphylococcalnuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-VI-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

In some embodiments of any of the aspects, a detectable label is achemiluminescent label, including, but not limited to lucigenin,luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole,acridinium salt and oxalate ester.

In some embodiments of any of the aspects, a detectable label can be aspectral colorimetric label including, but not limited to colloidal goldor colored glass or plastic (e.g., polystyrene, polypropylene, andlatex) beads.

In some embodiments of any of the aspects, antibodies can also belabeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5,HIS, or biotin. Other detection systems can also be used, for example, abiotin-streptavidin system. In this system, the antibodiesimmunoreactive (i. e. specific for) with the biomarker of interest isbiotinylated. Quantity of biotinylated antibody bound to the biomarkeris determined using a streptavidin-peroxidase conjugate and achromagenic substrate. Such streptavidin peroxidase detection kits arecommercially available, e. g. from DAKO; Carpinteria, Calif.

An antibody reagent can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the antibody reagent using such metalchelating groups as diethylenetriaminepentaacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

The methods as described herein can relate to determining if a subjecthas an increased level of CHI3L1 relative to a reference level. In someembodiments of any of the aspects, the reference level of CHI3L1 can bethe level of CHI3L1 in a healthy subject not having, or not diagnosed ashaving, e.g., cancer. In some embodiments of any of the aspects, thereference level can be the level in a sample of similar cell type,sample type, sample processing, and/or obtained from a subject ofsimilar age, sex and other demographic parameters as the sample/subjectfor which the level of CHI3L1 is to be determined. In some embodimentsof any of the aspects, the test sample and control reference sample areof the same type, that is, obtained from the same biological source, andcomprising the same composition, e.g., the same number and type of cellsand/or type of sample material. Accordingly, In some embodiments of anyof the aspects, the level of CHI3L1 which is increased can vary asdemographic factors such as age, gender, genotype, environmentalfactors, and individual medical histories vary. In some embodiments ofany of the aspects, the reference level can comprise the level of CHI3L1(e.g., CHI3L1 polypeptide) in a sample of the same type taken from asubject not exhibiting any signs or symptoms of, e.g., cancer. In someembodiments of any of the aspects, the reference expression level ofCHI3L1 can be the expression level of CHI3L1 in a prior sample obtainedfrom the subject. This permits a direct analysis of any change in levelsin that individual.

In some embodiments of any of the aspects, a level of CHI3L1 can beincreased relative to a reference level if the level of CHI3L1 is atleast 1.25× the reference level, e.g., at least 1.25×, at least 1.5×, atleast 2×, at least 3×, at least 4×, at least 5×, at least 6×, or greaterof the reference level. In some embodiments of any of the aspects, theexpression level of CHI3L1 can be normalized relative to the expressionlevel of one or more reference genes or reference proteins. In someembodiments of any of the aspects, the expression level of CHI3L1 can benormalized relative to a reference value.

In some embodiments of any of the aspects, the expression level of nomore than 20 other genes is determined. In some embodiments of any ofthe aspects, the expression level of no more than 10 other genes isdetermined.

The term “sample” or “test sample” as used herein denotes a sample takenor isolated from an organism, e.g., a urine sample from a subject.Exemplary biological samples include, but are not limited to, a biofluidsample; serum; plasma; urine; saliva; and/or tumor sample, etc. The termalso includes a mixture of the above-mentioned samples. The term “testsample” also includes untreated or pretreated (or pre-processed)biological samples. In some embodiments of any of the aspects, a testsample can comprise cells from a subject. As used herein, the term“biofluid” refers to any fluid obtained from a biological source andincludes, but is not limited to, blood, urine, and bodily secretions.

The test sample can be obtained by removing a sample from a subject, butcan also be accomplished by using a previously isolated sample (e.g.,isolated at a prior timepoint and isolated by the same or anotherperson). In addition, the test sample can be freshly collected or apreviously collected sample.

In some embodiments of any of the aspects, the test sample can be anuntreated test sample. As used herein, the phrase “untreated testsample” refers to a test sample that has not had any prior samplepre-treatment except for dilution and/or suspension in a solution.Exemplary methods for treating a test sample include, but are notlimited to, centrifugation, filtration, sonication, homogenization,heating, freezing and thawing, and combinations thereof. In someembodiments of any of the aspects, the test sample can be a frozen testsample, e.g., a frozen tissue. The frozen sample can be thawed beforeemploying methods, assays and systems described herein. After thawing, afrozen sample can be centrifuged before being subjected to methods,assays and systems described herein. In some embodiments of any of theaspects, the test sample is a clarified test sample, for example,prepared by centrifugation and collection of a supernatant comprisingthe clarified test sample. In some embodiments of any of the aspects, atest sample can be a pre-processed test sample, for example, supernatantor filtrate resulting from a treatment selected from the groupconsisting of centrifugation, filtration, thawing, purification, and anycombinations thereof. In some embodiments of any of the aspects, thetest sample can be treated with a chemical and/or biological reagent.Chemical and/or biological reagents can be employed to protect and/ormaintain the stability of the sample, including biomolecules (e.g.,nucleic acid and protein) therein, during processing. One exemplaryreagent is a protease inhibitor, which is generally used to protect ormaintain the stability of protein during processing. The skilled artisanis well aware of methods and processes appropriate for pre-processing ofbiological samples required for determination of the level of CHI3L1 asdescribed herein.

In some embodiments of any of the aspects, the methods, assays, andsystems described herein can further comprise a step of obtaining a testsample from a subject. In some embodiments of any of the aspects, thesubject can be a human subject.

In some embodiments of any of the aspects, the methods, assays, andsystems described herein can comprise creating a report based on thelevel of CHI3L1. In some embodiments of any of the aspects, the reportdenotes raw values for CHI3L1 in the test sample (plus, optionally, thelevel of CHI3L1 in a reference sample) or it indicates a percentage orfold increase in CHI3L1 as compared to a reference level, and/orprovides a signal that the subject is at risk of having, or not havingcancer.

As used herein “at risk of having” refers to at least a 2-fold greaterlikelihood of having a particular condition as compared to a subjectthat did not have an elevated and/or increased level of CHI3L1, e.g., a2-fold, or 2.5-fold, or 3-fold, or 4-fold, or greater risk.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments of any of the aspects, “reduce,” “reduction” or “decrease”or “inhibit” typically means a decrease by at least 10% as compared to areference level (e.g. the absence of a given treatment or agent) and caninclude, for example, a decrease by at least about 10%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, ormore. As used herein, “reduction” or “inhibition” does not encompass acomplete inhibition or reduction as compared to a reference level.“Complete inhibition” is a 100% inhibition as compared to a referencelevel. A decrease can be preferably down to a level accepted as withinthe range of normal for an individual without a given disorder.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments of any of the aspects, the terms “increased”, “increase”,“enhance”, or “activate” can mean an increase of at least 10% ascompared to a reference level, for example an increase of at least about20%, or at least about 30%, or at least about 40%, or at least about50%, or at least about 60%, or at least about 70%, or at least about80%, or at least about 90% or up to and including a 100% increase or anyincrease between 10-100% as compared to a reference level, or at leastabout a 2-fold, or at least about a 3-fold, or at least about a 4-fold,or at least about a 5-fold or at least about a 10-fold increase, or anyincrease between 2-fold and 10-fold or greater as compared to areference level. In the context of a marker or symptom, a “increase” isa statistically significant increase in such level.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids, including modifiedamino acids (e.g., phosphorylated, glycated, glycosylated, etc.) andamino acid analogs, regardless of its size or function. “Protein” and“polypeptide” are often used in reference to relatively largepolypeptides, whereas the term “peptide” is often used in reference tosmall polypeptides, but usage of these terms in the art overlaps. Theterms “protein” and “polypeptide” are used interchangeably herein whenreferring to a gene product and fragments thereof. Thus, exemplarypolypeptides or proteins include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, fragments, and analogs of the foregoing.

In the various embodiments described herein, it is further contemplatedthat variants (naturally occurring or otherwise), alleles, homologs,conservatively modified variants, and/or conservative substitutionvariants of any of the particular polypeptides described areencompassed. As to amino acid sequences, one of skill will recognizethat individual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters a single aminoacid or a small percentage of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid andretains the desired activity of the polypeptide. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles consistent with thedisclosure.

In some embodiments of any of the aspects, the polypeptide describedherein (or a nucleic acid encoding such a polypeptide) can be afunctional fragment of one of the amino acid sequences described herein.As used herein, a “functional fragment” is a fragment or segment of apeptide which retains at least 50% of the wildtype referencepolypeptide's activity according to the assays described below herein. Afunctional fragment can comprise conservative substitutions of thesequences disclosed herein.

In some embodiments of any of the aspects, the polypeptide describedherein can be a variant of a sequence described herein. In someembodiments of any of the aspects, the variant is a conservativelymodified variant. Conservative substitution variants can be obtained bymutations of native nucleotide sequences, for example. A “variant,” asreferred to herein, is a polypeptide substantially homologous to anative or reference polypeptide, but which has an amino acid sequencedifferent from that of the native or reference polypeptide because ofone or a plurality of deletions, insertions or substitutions. Variantpolypeptide-encoding DNA sequences encompass sequences that comprise oneor more additions, deletions, or substitutions of nucleotides whencompared to a native or reference DNA sequence, but that encode avariant protein or fragment thereof that retains activity. A widevariety of PCR-based site-specific mutagenesis approaches are known inthe art and can be applied by the ordinarily skilled artisan.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid or an analog thereof.The nucleic acid can be either single-stranded or double-stranded. Asingle-stranded nucleic acid can be one nucleic acid strand of adenatured double-stranded DNA. Alternatively, it can be asingle-stranded nucleic acid not derived from any double-stranded DNA.In one aspect, the nucleic acid can be DNA. In another aspect, thenucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA orcDNA. Suitable RNA can include, e.g., mRNA. In some embodiments of anyof the aspects, the nucleic acid can be a cDNA, e.g., a nucleic acidlacking introns.

Nucleic acid molecules encoding amino acid sequence variants ofantibodies or other polypeptides described herein are prepared by avariety of methods known in the art. These methods include, but are notlimited to preparation by oligonucleotide-mediated (or site-directed)mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlierprepared variant or a non-variant version of the antibody. A nucleicacid sequence encoding at least one antibody, portion or polypeptide asdescribed herein can be recombined with vector DNA in accordance withconventional techniques, including blunt-ended or staggered-endedtermini for ligation, restriction enzyme digestion to provideappropriate termini, filling in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andligation with appropriate ligases. Techniques for such manipulations canbe used to construct nucleic acid sequences which encode, e.g., amonoclonal antibody molecule, antibody reagent, antigen binding regionthereof, or CAR.

A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene expression as peptides orantibody portions in recoverable amounts. The precise nature of theregulatory regions needed for gene expression may vary from organism toorganism, as is well known in the analogous art.

In some embodiments of any of the aspects, a nucleic acid encoding,e.g., an antibody, antibody reagent, antigen-binding portion thereof,CAR, ligand, or inhibitory nucleic acid as described herein, iscomprised by a vector. In some of the aspects described herein, anucleic acid sequence encoding a polypeptide or nucleic acid asdescribed herein, or any module thereof, is operably linked to a vector.The term “vector”, as used herein, refers to a nucleic acid constructdesigned for delivery to a host cell or for transfer between differenthost cells. As used herein, a vector can be viral or non-viral. The term“vector” encompasses any genetic element that is capable of replicationwhen associated with the proper control elements and that can transfergene sequences to cells. A vector can include, but is not limited to, acloning vector, an expression vector, a plasmid, phage, transposon,cosmid, chromosome, virus, virion, etc.

As used herein, the term “expression vector” refers to a vector thatdirects expression of an RNA or polypeptide from sequences linked totranscriptional regulatory sequences on the vector. The sequencesexpressed will often, but not necessarily, be heterologous to the cell.An expression vector may comprise additional elements, for example, theexpression vector may have two replication systems, thus allowing it tobe maintained in two organisms, for example in human cells forexpression and in a prokaryotic host for cloning and amplification. Theterm “expression” refers to the cellular processes involved in producingRNA and proteins and as appropriate, secreting proteins, including whereapplicable, but not limited to, for example, transcription, transcriptprocessing, translation and protein folding, modification andprocessing. “Expression products” include RNA transcribed from a gene,and polypeptides obtained by translation of mRNA transcribed from agene. The term “gene” means the nucleic acid sequence which istranscribed (DNA) to RNA in vitro or in vivo when operably linked toappropriate regulatory sequences. The gene may or may not includeregions preceding and following the coding region, e.g., 5′ untranslated(5′UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as wellas intervening sequences (introns) between individual coding segments(exons).

As used herein, the term “viral vector” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle. The viral vectorcan contain the nucleic acid encoding a polypeptide or nucleic acid asdescribed herein in place of non-essential viral genes. The vectorand/or particle may be utilized for the purpose of transferring anynucleic acids into cells either in vitro or in vivo. Numerous forms ofviral vectors are known in the art.

By “recombinant vector” is meant a vector that includes a heterologousnucleic acid sequence, or “transgene” that is capable of expression invivo. It should be understood that the vectors described herein can, Insome embodiments of any of the aspects, be combined with other suitablecompositions and therapies. In some embodiments of any of the aspects,the vector is episomal. The use of a suitable episomal vector provides ameans of maintaining the nucleotide of interest in the subject in highcopy number extra chromosomal DNA thereby eliminating potential effectsof chromosomal integration.

In one aspect of any of the embodiments, described herein is a cellcomprising a polypeptide or nucleic acid as described herein, or anucleic acid encoding such a polypeptide or nucleic acid.

The expression of a polypeptide or nucleci acid as described herein canoccur in either prokaryotic or eukaryotic cells. Suitable hosts includebacterial or eukaryotic hosts, including yeast, insects, fungi, bird andmammalian cells either in vivo, or in situ, or host cells of mammalian,insect, bird or yeast origin. The mammalian cell or tissue can be ofhuman, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat,dog or cat origin, but any other mammalian cell may be used. Further, byuse of, for example, the yeast ubiquitin hydrolase system, in vivosynthesis of ubiquitin-transmembrane polypeptide fusion proteins can beaccomplished. The fusion proteins so produced can be processed in vivoor purified and processed in vitro, allowing synthesis of an antibody orportion thereof as described herein with a specified amino terminussequence. Moreover, problems associated with retention of initiationcodon-derived methionine residues in direct yeast (or bacterial)expression maybe avoided. Any of a series of yeast gene expressionsystems incorporating promoter and termination elements from theactively expressed genes coding for glycolytic enzymes produced in largequantities when yeast are grown in mediums rich in glucose can beutilized to obtain recombinant antibodies or antigen-binding portionsthereof as described herein. Known glycolytic genes can also providevery efficient transcriptional control signals. For example, thepromoter and terminator signals of the phosphoglycerate kinase gene canbe utilized.

Production of antibodies or antigen-binding portions thereof asdescribed herein in insects can be achieved. For example, by infectingthe insect host with a baculovirus engineered to express a transmembranepolypeptide by methods known to those of ordinary skill in the art.

In some embodiments of any of the aspects, the introduced nucleotidesequence is incorporated into a plasmid or viral vector capable ofautonomous replication in the recipient host. Any of a wide variety ofvectors can be employed for this purpose and are known and available tothose or ordinary skill in the art. Factors of importance in selecting aparticular plasmid or viral vector include: the ease with whichrecipient cells that contain the vector may be recognized and selectedfrom those recipient cells which do not contain the vector; the numberof copies of the vector which are desired in a particular host; andwhether it is desirable to be able to “shuttle” the vector between hostcells of different species.

Example prokaryotic vectors known in the art include plasmids such asthose capable of replication in E. coli., for example. Other geneexpression elements useful for the expression of cDNA include, but arenot limited to (a) viral transcription promoters and their enhancerelements, such as the SV40 early promoter, Rous sarcoma virus LTR, andMoloney murine leukemia virus; (b) splice regions and polyadenylationsites such as those derived from the SV40 late region, and (c)polyadenylation sites such as in SV40. Immunoglobulin cDNA genes can beexpressed, e.g., using as expression elements the SV40 early promoterand its enhancer, the mouse immunoglobulin H chain promoter enhancers,SV40 late region mRNA splicing, rabbit S-globin intervening sequence,immunoglobulin and rabbit S-globin polyadenylation sites, and SV40polyadenylation elements.

For immunoglobulin genes comprised of part cDNA, part genomic DNA, thetranscriptional promoter can be human cytomegalovirus, the promoterenhancers can be cytomegalovirus and mouse/human immunoglobulin, andmRNA splicing and polyadenylation regions can be the native chromosomalimmunoglobulin sequences.

In some embodiments of any of the aspects, for expression of cDNA genesin rodent cells, the transcriptional promoter is a viral LTR sequence,the transcriptional promoter enhancers are either or both the mouseimmunoglobulin heavy chain enhancer and the viral LTR enhancer, thesplice region contains an intron of greater than 31 bp, and thepolyadenylation and transcription termination regions are derived fromthe native chromosomal sequence corresponding to the immunoglobulinchain being synthesized. In other embodiments, cDNA sequences encodingother proteins are combined with the above-recited expression elementsto achieve expression of the proteins in mammalian cells.

A gene is assembled in, or inserted into, an expression vector.Recipient cells capable of expressing the chimeric immunoglobulin chaingene product are then transfected singly with an antibody,antigen-binding portion thereof, or CAR, or chimeric H or chimeric Lchain-encoding gene, or are co-transfected with a chimeric H and achimeric L chain gene. The transfected recipient cells are culturedunder conditions that permit expression of the incorporated genes andthe expressed immunoglobulin chains or intact antibodies or fragmentsare recovered from the culture.

In some embodiments of any of the aspects, the genes encoding thepolypeptide or nucleic acid are assembled in separate expression vectorsthat are then used to co-transfect a recipient cell. Each vector cancontain two selectable genes, a first selectable gene designed forselection in a bacterial system and a second selectable gene designedfor selection in a eukaryotic system, wherein each vector has adifferent pair of genes. This strategy results in vectors which firstdirect the production, and permit amplification, of the genes in abacterial system. The genes so produced and amplified in a bacterialhost are subsequently used to co-transfect a eukaryotic cell, and allowselection of a co-transfected cell carrying the desired transfectedgenes. Non-limiting examples of selectable genes for use in a bacterialsystem are the gene that confers resistance to ampicillin and the genethat confers resistance to chloramphenicol. Selectable genes for use ineukaryotic transfectants include the xanthine guanine phosphoribosyltransferase gene (designated gpt) and the phosphotransferase gene fromTn5 (designated neo). Alternatively the genes can be assembled on thesame expression vector.

For transfection of the expression vectors and production of thepolypeptide or nucleic acids described herein, the recipient cell linecan be a myeloma cell. Myeloma cells can synthesize, assemble andsecrete immunoglobulins encoded by transfected immunoglobulin genes andpossess the mechanism for glycosylation of the immunoglobulin. Forexample, In some embodiments of any of the aspects, the recipient cellis the recombinant Ig-producing myeloma cell SP2/0 (ATCC #CRL 8287).SP2/0 cells produce only immunoglobulin encoded by the transfectedgenes. Myeloma cells can be grown in culture or in the peritoneal cavityof a mouse, where secreted immunoglobulin can be obtained from ascitesfluid. Other suitable recipient cells include lymphoid cells such as Blymphocytes of human or non-human origin, hybridoma cells of human ornon-human origin, or interspecies heterohybridoma cells.

An expression vector carrying a polypeptide or nucleic acid as describedherein can be introduced into an appropriate host cell by any of avariety of suitable means, including such biochemical means astransformation, transfection, conjugation, protoplast fusion, calciumphosphate-precipitation, and application with polycations such asdiethylaminoethyl (DEAE) dextran, and such mechanical means aselectroporation, direct microinjection, and microprojectile bombardment,as known to one of ordinary skill in the art.

Traditionally, monoclonal antibodies have been produced as nativemolecules in murine hybridoma lines. In addition to that technology, themethods and compositions described herein provide for recombinant DNAexpression of monoclonal antibodies. This allows the production ofhumanized antibodies as well as a spectrum of antibody derivatives andfusion proteins in a host species of choice. The production ofantibodies in bacteria, yeast, transgenic animals and chicken eggs arealso alternatives for hybridoma-based production systems. The mainadvantages of transgenic animals are potential high yields fromrenewable sources.

In one aspect, a cell comprising an isolated antibody, antigen-bindingportion thereof, or CAR as described herein is provided. In someembodiments of any of the aspects, the isolated antibody,antigen-binding portion thereof, or CAR as described herein is expressedon the cell surface. In some embodiments of any of the aspects, the cellcomprises a nucleic acid encoding an isolated antibody, antigen-bindingportion thereof, or CAR as described herein.

In some embodiments of any of the aspects, the cell is an immune cell.As used herein, “immune cell” refers to a cell that plays a role in theimmune response. Immune cells are of hematopoietic origin, and includelymphocytes, such as B cells and T cells; natural killer cells; myeloidcells, such as monocytes, macrophages, eosinophils, mast cells,basophils, and granulocytes. In some embodiments of any of the aspects,the cell is a T cell; a NK cell; a NKT cell; lymphocytes, such as Bcells and T cells; and myeloid cells, such as monocytes, macrophages,cosinophils, mast cells, basophils, and granulocytes.

In particular embodiments, a cell (e.g., an immune cell) is transducedwith a retroviral vector, e.g., a lentiviral vector, encoding a CAR. Forexample, an immune effector cell is transduced with a vector encoding aCAR that comprises an anti-CHI3L1 antibody or antigen binding portionthereof that binds a CHI3L1 polypeptide, with an intracellular signalingdomain of CD3ζ, CD28, 4-1BB, Ox40, or any combinations thereof. Thus,these transduced cells can elicit a CAR-mediated cytotoxic response.

Retroviruses are a common tool for gene delivery. In particularembodiments, a retrovirus is used to deliver a polynucleotide encoding achimeric antigen receptor (CAR) to a cell. As used herein, the term“retrovirus” refers to an RNA virus that reverse transcribes its genomicRNA into a linear double-stranded DNA copy and subsequently covalentlyintegrates its genomic DNA into a host genome. Once the virus isintegrated into the host genome, it is referred to as a “provirus.” Theprovirus serves as a template for RNA polymerase II and directs theexpression of RNA molecules which encode the structural proteins andenzymes needed to produce new viral particles.

Illustrative retroviruses suitable for use in particular embodiments,include, but are not limited to: Moloney murine leukemia virus (M-MuLV),Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus(GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemiavirus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) andlentivirus.

As used herein, the term “lentivirus” refers to a group (or genus) ofcomplex retroviruses. Illustrative lentiviruses include, but are notlimited to: HIV (human immunodeficiency virus; including HIV type 1, andHIV type 2); visna-maedi virus (VMV) virus; the caprinearthritis-encephalitis virus (CAEV); equine infectious anemia virus(EIAV); feline immunodeficiency virus (FIV); bovine immune deficiencyvirus (BIV); and simian immunodeficiency virus (SIV). In one embodiment,HIV based vector backbones (i.e., HIV cis-acting sequence elements) arepreferred. In particular embodiments, a lentivirus is used to deliver apolynucleotide comprising a CAR to a cell.

Retroviral vectors and more particularly lentiviral vectors may be usedin practicing particular embodiments of the present invention.Accordingly, the term “retrovirus” or “retroviral vector”, as usedherein is meant to include “lentivirus” and “lentiviral vectors”respectively.

In some embodiments of any of the aspects, the agent that inhibits agiven target is an inhibitory nucleic acid. In some embodiments of anyof the aspects, inhibitors of the expression of a given gene can be aninhibitory nucleic acid. As used herein, “inhibitory nucleic acid”refers to a nucleic acid molecule which can inhibit the expression of atarget, e.g., double-stranded RNAs (dsRNAs), inhibitory RNAs (iRNAs),miRNAs, amiRNAs, and the like.

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). The inhibitory nucleic acids described herein caninclude an RNA strand (the antisense strand) having a region which is 30nucleotides or less in length, i.e., 15-30 nucleotides in length,generally 19-24 nucleotides in length, which region is substantiallycomplementary to at least part the targeted mRNA transcript. The use ofthese iRNAs enables the targeted degradation of mRNA transcripts,resulting in decreased expression and/or activity of the target.

As used herein, the term “iRNA” refers to an agent that contains RNA (ormodified nucleic acids as described below herein) and which mediates thetargeted cleavage of an RNA transcript via an RNA-induced silencingcomplex (RISC) pathway. In some embodiments of any of the aspects, aniRNA as described herein effects inhibition of the expression and/ortranslation and/or activity of a target, e.g. CHI3L1. In someembodiments of any of the aspects, contacting a cell with the inhibitor(e.g. an iRNA) results in a decrease in the target mRNA level in a cellby at least about 5%, about 10%, about 20%, about 30%, about 40%, about50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%,up to and including 100% of the target mRNA level found in the cellwithout the presence of the iRNA. In some embodiments of any of theaspects, administering an inhibitor (e.g. an iRNA) to a subject resultsin a decrease in the target mRNA level in the subject by at least about5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, about 90%, about 95%, about 99%, up to andincluding 100% of the target mRNA level found in the subject without thepresence of the iRNA.

In some embodiments of any of the aspects, the iRNA can be a dsRNA. AdsRNA includes two RNA strands that are sufficiently complementary tohybridize to form a duplex structure under conditions in which the dsRNAwill be used. One strand of a dsRNA (the antisense strand) includes aregion of complementarity that is substantially complementary, andgenerally fully complementary, to a target sequence. The target sequencecan be derived from the sequence of an mRNA formed during the expressionof the target, e.g., it can span one or more intron boundaries. Theother strand (the sense strand) includes a region that is complementaryto the antisense strand, such that the two strands hybridize and form aduplex structure when combined under suitable conditions. Generally, theduplex structure is between 15 and 30 base pairs in length inclusive,more generally between 18 and 25 base pairs in length inclusive, yetmore generally between 19 and 24 base pairs in length inclusive, andmost generally between 19 and 21 base pairs in length, inclusive.Similarly, the region of complementarity to the target sequence isbetween 15 and 30 base pairs in length inclusive, more generally between18 and 25 base pairs in length inclusive, yet more generally between 19and 24 base pairs in length inclusive, and most generally between 19 and21 base pairs in length nucleotides in length, inclusive. In someembodiments of any of the aspects, the dsRNA is between 15 and 20nucleotides in length, inclusive, and in other embodiments, the dsRNA isbetween 25 and 30 nucleotides in length, inclusive. As the ordinarilyskilled person will recognize, the targeted region of an RNA targetedfor cleavage will most often be part of a larger RNA molecule, often anmRNA molecule. Where relevant, a “part” of an mRNA target is acontiguous sequence of an mRNA target of sufficient length to be asubstrate for RNAi-directed cleavage (i.e., cleavage through a RISCpathway). dsRNAs having duplexes as short as 9 base pairs can, undersome circumstances, mediate RNAi-directed RNA cleavage. Most often atarget will be at least 15 nucleotides in length, preferably 15-30nucleotides in length.

Exemplary embodiments of types of inhibitory nucleic acids can include,e.g., siRNA, shRNA, miRNA, and/or amiRNA, which are well known in theart.

In some embodiments of any of the aspects, the RNA of an iRNA, e.g., adsRNA, is chemically modified to enhance stability or other beneficialcharacteristics. The nucleic acids described herein may be synthesizedand/or modified by methods well established in the art, such as thosedescribed in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, whichis hereby incorporated herein by reference. Modifications include, forexample, (a) end modifications, e.g., 5′ end modifications(phosphorylation, conjugation, inverted linkages, etc.) 3′ endmodifications (conjugation, DNA nucleotides, inverted linkages, etc.),(b) base modifications, e.g., replacement with stabilizing bases,destabilizing bases, or bases that base pair with an expanded repertoireof partners, removal of bases (abasic nucleotides), or conjugated bases,(c) sugar modifications (e.g., at the 2′ position or 4′ position) orreplacement of the sugar, as well as (d) backbone modifications,including modification or replacement of the phosphodiester linkages.Specific examples of RNA compounds useful in the embodiments describedherein include, but are not limited to RNAs containing modifiedbackbones or no natural internucleoside linkages. RNAs having modifiedbackbones include, among others, those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified RNAs that do not have aphosphorus atom in their internucleoside backbone can also be consideredto be oligonucleosides. In some embodiments of any of the aspects, themodified RNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones can include, for example, phosphorothioates,chiral phosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included. Modified RNAbackbones that do not include a phosphorus atom therein have backbonesthat are formed by short chain alkyl or cycloalkyl internucleosidelinkages, mixed heteroatoms and alkyl or cycloalkyl internucleosidelinkages, or one or more short chain heteroatomic or heterocyclicinternucleoside linkages. These include those having morpholino linkages(formed in part from the sugar portion of a nucleoside); siloxanebackbones; sulfide, sulfoxide and sulfone backbones; formacetyl andthioformacetyl backbones; methylene formacetyl and thioformacetylbackbones; alkene containing backbones; sulfamate backbones;methyleneimino and methylenehydrazino backbones; sulfonate andsulfonamide backbones; amide backbones; others having mixed N, O, S andCH2 component parts, and oligonucleosides with heteroatom backbones, andin particular —CH2-NH—CH2-, —CH2-N(CH3)-O—CH2-[known as a methylene(methylimino) or MMI backbone], —CH2-O—N(CH3)-CH2-,—CH2-N(CH3)-N(CH3)-CH2- and —N(CH3)-CH2-CH2-[wherein the nativephosphodiester backbone is represented as —O—P—O—CH2-].

In other RNA mimetics suitable or contemplated for use in iRNAs, boththe sugar and the internucleoside linkage, i.e., the backbone, of thenucleotide units are replaced with novel groups. The base units aremaintained for hybridization with an appropriate nucleic acid targetcompound. One such oligomeric compound, an RNA mimetic that has beenshown to have excellent hybridization properties, is referred to as apeptide nucleic acid (PNA). In PNA compounds, the sugar backbone of anRNA is replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone.

The RNA of an iRNA can also be modified to include one or more lockednucleic acids (LNA). A locked nucleic acid is a nucleotide having amodified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193).

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, described herein can include one of thefollowing at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkylor C2 to C10 alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3,O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 toabout 10. In some embodiments of any of the aspects, dsRNAs include oneof the following at the 2′ position: C1 to C10 lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN,Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments of any of the aspects, the modification includes a 2′methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., analkoxy-alkoxy group. Another exemplary modification is2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as2′-DMAOE, as described in examples herein below, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH2-O—CH2-N(CH2)2, also described in examples herein below.

Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy(2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar.

An inhibitory nucleic acid can also include nucleobase (often referredto in the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo,particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and3-deazaadenine. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the inhibitory nucleic acids featuredin the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds., dsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are exemplary base substitutions, evenmore particularly when combined with 2′-O-methoxyethyl sugarmodifications.

The preparation of the modified nucleic acids, backbones, andnucleobases described above are well known in the art.

Another modification of an inhibitory nucleic acid featured in theinvention involves chemically linking to the inhibitory nucleic acid toone or more ligands, moieties or conjugates that enhance the activity,cellular distribution, pharmacokinetic properties, or cellular uptake ofthe iRNA. Such moieties include but are not limited to lipid moietiessuch as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci.USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med.Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan etal., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990,259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), aphospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.,1990, 18:3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In some embodiments of any of the aspects, a polypeptide, nucleic acid,or cell as described herein can be engineered. As used herein,“engineered” refers to the aspect of having been manipulated by the handof man. For example, a polypeptide is considered to be “engineered” whenat least one aspect of the polypeptide, e.g., its sequence, has beenmanipulated by the hand of man to differ from the aspect as it exists innature. As is common practice and is understood by those in the art,progeny of an engineered cell are typically still referred to as“engineered” even though the actual manipulation was performed on aprior entity.

In some embodiments of any of the aspects, a nucleic acid encoding apolypeptide as described herein (e.g. an antibody or antibody reagent)is comprised by a vector. In some of the aspects described herein, anucleic acid sequence encoding a given polypeptide as described herein,or any module thereof, is operably linked to a vector. A vector caninclude, but is not limited to, a cloning vector, an expression vector,a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.

As used herein, the term “expression vector” refers to a vector thatdirects expression of an RNA or polypeptide from sequences linked totranscriptional regulatory sequences on the vector. The sequencesexpressed will often, but not necessarily, be heterologous to the cell.An expression vector may comprise additional elements, for example, theexpression vector may have two replication systems, thus allowing it tobe maintained in two organisms, for example in human cells forexpression and in a prokaryotic host for cloning and amplification. Theterm “expression” refers to the cellular processes involved in producingRNA and proteins and as appropriate, secreting proteins, including whereapplicable, but not limited to, for example, transcription, transcriptprocessing, translation and protein folding, modification andprocessing. “Expression products” include RNA transcribed from a gene,and polypeptides obtained by translation of mRNA transcribed from agene. The term “gene” means the nucleic acid sequence which istranscribed (DNA) to RNA in vitro or in vivo when operably linked toappropriate regulatory sequences. The gene may or may not includeregions preceding and following the coding region, e.g. 5′ untranslated(5′UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as wellas intervening sequences (introns) between individual coding segments(exons).

The term “isolated” or “partially purified” as used herein refers, inthe case of a nucleic acid or polypeptide, to a nucleic acid orpolypeptide separated from at least one other component (e.g., nucleicacid or polypeptide) that is present with the nucleic acid orpolypeptide as found in its natural source and/or that would be presentwith the nucleic acid or polypeptide when expressed by a cell, orsecreted in the case of secreted polypeptides. A chemically synthesizednucleic acid or polypeptide or one synthesized using in vitrotranscription/translation is considered “isolated.” The terms “purified”or “substantially purified” refer to an isolated nucleic acid orpolypeptide that is at least 95% by weight the subject nucleic acid orpolypeptide, including, for example, at least 96%, at least 97%, atleast 98%, at least 99% or more. In some embodiments of any of theaspects, the antibody, antigen-binding portion thereof, or CAR describedherein is isolated. In some embodiments of any of the aspects, theantibody, antibody reagent, antigen-binding portion thereof, or CARdescribed herein is purified.

As used herein, “engineered” refers to the aspect of having beenmanipulated by the hand of man. For example, an antibody, antibodyreagent, antigen-binding portion thereof, or CAR is considered to be“engineered” when the sequence of the antibody, antibody reagent,antigen-binding portion thereof, or CAR is manipulated by the hand ofman to differ from the sequence of an antibody as it exists in nature.As is common practice and is understood by those in the art, progeny andcopies of an engineered polynucleotide and/or polypeptide are typicallystill referred to as “engineered” even though the actual manipulationwas performed on a prior entity.

As used herein, an “epitope” can be formed on a polypeptide both fromcontiguous amino acids, or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents, whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5, about 9, or about 8-10 amino acids in a uniquespatial conformation. An “epitope” includes the unit of structureconventionally bound by an immunoglobulin VH/VL pair. Epitopes definethe minimum binding site for an antibody, and thus represent the targetof specificity of an antibody. In the case of a single domain antibody,an epitope represents the unit of structure bound by a variable domainin isolation. The terms “antigenic determinant” and “epitope” can alsobe used interchangeably herein. In certain embodiments, epitopedeterminants include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and/or specific charge characteristics.

“Avidity” is the measure of the strength of binding between anantigen-binding molecule (such as an antibody or antigen-binding portionthereof described herein) and the pertinent antigen. Avidity is relatedto both the affinity between an antigenic determinant and its antigenbinding site on the antigen-binding molecule, and the number ofpertinent binding sites present on the antigen-binding molecule.Typically, antigen-binding proteins (such as an antibody or portion ofan antibody as described herein) will bind to their cognate or specificantigen with a dissociation constant (KD of 10⁻⁵ to 10⁻¹² moles/liter orless, such as 10⁻⁷ to 10⁻¹² moles/liter or less, or 10⁻⁸ to 10⁻¹²moles/liter (i.e., with an association constant (KA) of 10⁵ to 10¹²liter/moles or more, such as 10⁷ to 10¹² liter/moles or 10⁸ to 10¹²liter/moles). Any KD value greater than 10⁻⁴ mol/liter (or any KA valuelower than 10⁴ M⁻¹) is generally considered to indicate non-specificbinding. The KD for biological interactions which are consideredmeaningful (e.g., specific) are typically in the range of 10⁻¹⁰ M (0.1nM) to 10⁻⁵ M (10000 nM). The stronger an interaction, the lower is itsKD. For example, a binding site on an antibody or portion thereofdescribed herein will bind to the desired antigen with an affinity lessthan 500 nM, such as less than 200 nM, or less than 10 nM, such as lessthan 500 pM. Specific binding of an antigen-binding protein to anantigen or antigenic determinant can be determined in any suitablemanner known per se, including, for example, Scatchard analysis and/orcompetitive binding assays, such as radioimmunoassays (RIA), enzymeimmunoassays (EIA) and sandwich competition assays, and the differentvariants thereof known per se in the art; as well as other techniques asmentioned herein.

Accordingly, as used herein, “selectively binds” or “specifically binds”refers to the ability of an peptide (e.g., an antibody, CAR or portionthereof) described herein to bind to a target, such as an antigenpresent on the cell-surface of a cancer cell, with a KD 10⁻⁵M (10000 nM)or less, e.g., 10⁻⁶M, 10⁻⁷M, 10⁻⁸ M, 10⁻⁹M, 10⁻¹⁰ M, 10⁻¹¹M, 10⁻¹²M, orless. Specific binding can be influenced by, for example, the affinityand avidity of the polypeptide agent and the concentration ofpolypeptide agent. The person of ordinary skill in the art can determineappropriate conditions under which the polypeptide agents describedherein selectively bind the targets using any suitable methods, such astitration of a polypeptide agent in a suitable cell binding assay. Apolypeptide specifically bound to a target is not displaced by anon-similar competitor. In certain embodiments, an antibody,antigen-binding portion thereof, or CAR is said to specifically bind anantigen when it preferentially recognizes its target antigen in acomplex mixture of proteins and/or macromolecules.

In some embodiments of any of the aspects, an antibody, antigen-bindingportion thereof, and/or CAR as described herein binds to CHI3L1 with adissociation constant (KD) of 10⁻⁵ M (10000 nM) or less, e.g., 10⁻⁶ M,10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹M, 10⁻¹² M, or less. In someembodiments of any of the aspects, an antibody, antigen-binding portionthereof, and/or CAR as described herein binds to CHI3L1 with adissociation constant (K_(D)) of from about 10⁻⁵ M to 10⁻⁶ M. In someembodiments of any of the aspects, an antibody, antigen-binding portionthereof, and/or CAR as described herein binds to CHI3L1 with adissociation constant (K_(D)) of from about 10⁻⁶ M to 10⁻⁷ M. In someembodiments of any of the aspects, an antibody, antigen-binding portionthereof, and/or CAR as described herein binds to CHI3L1 with adissociation constant (K_(D)) of from about 10⁻⁷ M to 10⁻⁸ M. In someembodiments of any of the aspects, an antibody, antigen-binding portionthereof, and/or CAR as described herein binds to CHI3L1 with adissociation constant (K_(D)) of from about 10⁻⁸ M to 10⁻⁹ M. In someembodiments of any of the aspects, an antibody, antigen-binding portionthereof, and/or CAR as described herein binds to CHI3L1 with adissociation constant (K_(D)) of from about 10⁻⁹ M to 10⁻¹⁰ M. In someembodiments of any of the aspects, an antibody, antigen-binding portionthereof, and/or CAR as described herein binds to CHI3L1 with adissociation constant (KD) of from about 10⁻¹⁰ M to 10⁻¹¹M. In someembodiments of any of the aspects, an antibody, antigen-binding portionthereof, and/or CAR as described herein binds to CHI3L1 with adissociation constant (KD) of from about 10⁻¹¹ M to 10⁻¹² M. In someembodiments of any of the aspects, an antibody, antigen-binding portionthereof, and/or CAR as described herein binds to CHI3L1 with adissociation constant (KD) of less than 10⁻¹² M.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject.

The term “agent” refers generally to any entity which is normally notpresent or not present at the levels being administered to a cell,tissue or subject. An agent can be selected from a group including butnot limited to: polynucleotides; polypeptides; small molecules; andantibodies or antigen-binding fragments thereof. A polynucleotide can beRNA or DNA, and can be single or double stranded, and can be selectedfrom a group including, for example, nucleic acids and nucleic acidanalogues that encode a polypeptide. A polypeptide can be, but is notlimited to, a naturally-occurring polypeptide, a mutated polypeptide ora fragment thereof that retains the function of interest. Furtherexamples of agents include, but are not limited to a nucleic acidaptamer, peptide-nucleic acid (PNA), locked nucleic acid (LNA), smallorganic or inorganic molecules; saccharide; oligosaccharides;polysaccharides; biological macromolecules, peptidomimetics; nucleicacid analogs and derivatives; extracts made from biological materialssuch as bacteria, plants, fungi, or mammalian cells or tissues andnaturally occurring or synthetic compositions. An agent can be appliedto the media, where it contacts the cell and induces its effects.Alternatively, an agent can be intracellular as a result of introductionof a nucleic acid sequence encoding the agent into the cell and itstranscription resulting in the production of the nucleic acid and/orprotein environmental stimuli within the cell. In some embodiments ofany of the aspects, the agent is any chemical, entity or moiety,including without limitation synthetic and naturally-occurringnon-proteinaceous entities. In certain embodiments the agent is a smallmolecule having a chemical moiety selected, for example, fromunsubstituted or substituted alkyl, aromatic, or heterocyclyl moietiesincluding macrolides, leptomycins and related natural products oranalogues thereof. Agents can be known to have a desired activity and/orproperty, or can be selected from a library of diverse compounds. Asused herein, the term “small molecule” can refer to compounds that are“natural product-like,” however, the term “small molecule” is notlimited to “natural product-like” compounds. Rather, a small molecule istypically characterized in that it contains several carbon-carbon bonds,and has a molecular weight more than about 50, but less than about 5000Daltons (5 kD). Preferably the small molecule has a molecular weight ofless than 3 kD, still more preferably less than 2 kD, and mostpreferably less than 1 kD. In some cases it is preferred that a smallmolecule have a molecular mass equal to or less than 700 Daltons.

The term “aptamer” refers to a nucleic acid molecule that is capable ofbinding to a target molecule, such as a polypeptide. For example, anaptamer of the invention can specifically bind to a target molecule, orto a molecule in a signaling pathway that modulates the expressionand/or activity of a target molecule. The generation and therapeutic useof aptamers are well established in the art. See, e.g., U.S. Pat. No.5,475,096.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in immunology andmolecular biology can be found in The Merck Manual of Diagnosis andTherapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011(ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), TheEncyclopedia of Molecular Cell Biology and Molecular Medicine, publishedby Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8);Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway'sImmunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor& Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's GenesXI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning: ALaboratory Manual, 4^(th) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., BasicMethods in Molecular Biology, Elsevier Science Publishing, Inc., NewYork, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology:DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); CurrentProtocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), JohnWiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocolsin Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons,Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan,ADA M Kruisbeck, David H Margulies, Ethan M Shevach, Warren Strobe,(eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737),the contents of which are all incorporated by reference herein in theirentireties.

One of skill in the art can readily identify a chemotherapeutic agent ofuse (e.g. see Physicians' Cancer Chemotherapy Drug Manual 2014, EdwardChu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles ofCancer Therapy, Chapter 85 in Harrison's Principles of InternalMedicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era ofMolecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 inAbeloff s Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): TheCancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

In some embodiments of any of the aspects, the disclosure describedherein does not concern a process for cloning human beings, processesfor modifying the germ line genetic identity of human beings, uses ofhuman embryos for industrial or commercial purposes or processes formodifying the genetic identity of animals which are likely to cause themsuffering without any substantial medical benefit to man or animal, andalso animals resulting from such processes.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   1. A method of treating cancer in a subject in need thereof, the    method comprising administering:    -   an inhibitor of CHI3L1; and    -   an inhibitor of an immune checkpoint protein.-   2. The method of paragraph 1, wherein the inhibitor of CHI3L1 is an    antibody, antibody reagent, antigen-binding fragment thereof, or CAR    that specifically binds to CHI3L1.-   3. The method of paragraph 2, wherein an antibody, antibody reagent,    antigen-binding fragment thereof, or CAR comprises at least one    heavy or light chain complementarity determining region (CDR)    selected from the group consisting of:    -   (a) a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 4;    -   (b) a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 5;    -   (c) a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 6;    -   (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 1;    -   (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 2; and    -   (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 3; or

a conservative substitution variant of one or more of (a)-(f).

-   4. The method of paragraph 3, wherein the antibody, antibody    reagent, antigen-binding portion thereof, or CAR of comprises heavy    chain CDRs having the amino acid sequences of SEQ ID NOs: 1-3 or a    conservative substitution variant of such amino acid sequence.-   5. The method of any of paragraphs 3-4, wherein the antibody,    antibody reagent, antigen-binding portion thereof or CAR comprises    light chain CDRs having the amino acid sequences of SEQ ID NOs: 4-6    or a conservative substitution variant of such amino acid sequence.-   6. The method of any of paragraphs 3-5, wherein the antibody,    antibody reagent, antigen-binding portion thereof or CAR comprises    light chain CDRs having the amino acid sequences of SEQ ID NOs: 4-6    and heavy chain CDRs having the amino acid sequences of SEQ ID NOs:    1-3 or a conservative substitution variant of such amino acid    sequence.-   7. The method of paragraph 2, wherein the inhibitor of CHI3L1 is    antibody, antibody reagent, antigen-binding portion thereof or CAR    that competes for binding to CHI3L1 with an antibody comprising    light chain CDRs having the amino acid sequences of SEQ ID NOs: 4-6    and heavy chain CDRs having the amino acid sequences of SEQ ID NOs:    1-3.-   8. The method of any of paragraphs 1-7, wherein the inhibitor of    CHI3L1 binds a CHI3L1 polypeptide at an epitope selected from SEQ ID    NOs: 13-24.-   9. The method of paragraph 1, wherein the inhibitor of CHI3L1 is an    inhibitor of a CHI3L1 receptor.-   10. The method of paragraph 9, wherein the CHI3L1 receptor is    IL13Rα2, TMEM219, or CRTH2.-   11. The method of any of paragraphs 9-10, wherein the inhibitor is    an inhibitory antibody or nucleic acid molecule.-   12. The method of any of paragraph 1-11, wherein the cancer is a    primary cancer or a metastatic cancer.-   13. The method of any of paragraphs 1-12, wherein the cancer is    malignant cancer.-   14. The method of any of paragraphs 1-13, wherein the cancer is    selected from the group consisting of:    -   prostate cancer, colon cancer, rectal cancer, ovarian cancer,        kidney cancer, breast cancer, glioblastoma, melanoma, malignant        melanoma, and lung cancer.-   15. The method of any of paragraphs 1-14, wherein the inhibitor of    an immune checkpoint protein is an antibody, antibody reagent,    antigen-binding fragment thereof, or CAR that specifically binds to    at least one immune checkpoint protein.-   16. The method of any of paragraphs 1-15, wherein the inhibitor of    an immune checkpoint protein is a natural ligand thereof-   17. The method of paragraph 16, wherein the natural ligand comprises    PD-L1 or B7.-   18. The method of any of paragraphs 1-17, wherein the immune    checkpoint protein is selected from the group consisting of:    -   PD-1; PD-L1; PD-L2; TIM-3; CTLA4; TIGTT; DD1-α; A2AR; B7-H3;        B7-H4; BTLA; IDO; TDO; KIR; and LAG3.-   19. The method of paragraph 18, wherein the inhibitor of an immune    checkpoint protein is selected from the group consisting of:    -   MGA271; ipilimumab; pembrolizumab; nivolumab; atezolizumab;        galiximab; IMP321; BMS-986016; SMB-663513; PF-05082566; IPH2101;        KW-0761; CDX-1127; MEDI-6769; CP-870,893; tremelimumab;        pidilizumab; MPDL3280A; MEDI4736; MSB0010718C; AUNP12; avelumab;        and durvalumab.-   20. The method of any of paragraphs 1-19, wherein the inhibitor of    CHI3L1 and the inhibitor of an immune checkpoint protein are present    in the same bivalent antibody reagent.-   21. The method of paragraph 20, wherein the immune checkpoint    protein is PD-1 or CTLA4.-   22. The method of any of paragraphs 1-21, wherein the subject is a    subject determined to have an elevated level of CHI3L1.-   23. The method of paragraph 22, wherein the CHI3L1 is circulating    CHI3L1.-   24. A pharmaceutical composition comprising    -   an inhibitor of CHI3L1; and    -   an inhibitor of an immune checkpoint protein.-   25. A kit comprising    -   a pharmaceutical composition comprising an inhibitor of CHI3L1;        and    -   a pharmaceutical composition comprising an inhibitor of an        immune checkpoint protein.-   26. The kit or composition of any of paragraphs 24-25, wherein the    inhibitor of CHI3L1 is an antibody, antibody reagent,    antigen-binding fragment thereof, or CAR that specifically binds to    CHI3L1.-   27. The kit or composition of paragraph 26, wherein an antibody,    antibody reagent, antigen-binding fragment thereof, or CAR comprises    at least one heavy or light chain complementarity determining region    (CDR) selected from the group consisting of:    -   (a) a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 4;    -   (b) a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 5;    -   (c) a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 6;    -   (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 1;    -   (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 2; and    -   (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 3; or

a conservative substitution variant of one or more of (a)-(f).

-   28. The kit or composition of any of paragraphs 26-27, wherein the    antibody, antibody reagent, antigen-binding portion thereof, or CAR    of comprises heavy chain CDRs having the amino acid sequences of SEQ    ID NOs: 1-3 or a conservative substitution variant of such amino    acid sequence.-   29. The kit or composition of any of paragraphs 26-28, wherein the    antibody, antibody reagent, antigen-binding portion thereof or CAR    comprises light chain CDRs having the amino acid sequences of SEQ ID    NOs: 4-6 or a conservative substitution variant of such amino acid    sequence.-   30. The kit or composition of any of paragraphs 26-29, wherein the    antibody, antibody reagent, antigen-binding portion thereof or CAR    comprises light chain CDRs having the amino acid sequences of SEQ ID    NOs: 4-6 and heavy chain CDRs having the amino acid sequences of SEQ    ID NOs: 1-3 or a conservative substitution variant of such amino    acid sequence.-   31. The kit or composition of any of paragraphs 24-25, wherein the    inhibitor of CHI3L1 is antibody, antibody reagent, antigen-binding    portion thereof or CAR that competes for binding to CHI3L1 with an    antibody comprising light chain CDRs having the amino acid sequences    of SEQ ID NOs: 4-6 and heavy chain CDRs having the amino acid    sequences of SEQ ID NOs: 1-3.-   32. The kit or composition of any of paragraphs 24-31, wherein the    inhibitor of CHI3L1 binds a CHI3L1 polypeptide at an epitope    selected from SEQ ID NOs: 13-24.-   33. The kit or composition of any of paragraphs 24-25, wherein the    inhibitor of CHI3L1 is an inhibitor of a CHI3L1 receptor.-   34. The kit or composition of paragraph 33, wherein the CHI3L1    receptor is IL13Rα2, TMEM219, or CRTH2.-   35. The kit or composition of any of paragraphs 33-34, wherein the    inhibitor is an inhibitory antibody or nucleic acid molecule.-   36. The kit or composition of any of paragraphs 24-35, wherein the    inhibitor of an immune checkpoint protein is an antibody, antibody    reagent, antigen-binding fragment thereof, or CAR that specifically    binds to at least one immune checkpoint protein.-   37. The kit or composition of any of paragraphs 24-36, wherein the    inhibitor of an immune checkpoint protein is a natural ligand    thereof.-   38. The kit or composition of paragraph 37, wherein the natural    ligand comprises PD-L1 or B7.-   39. The kit or composition of any of paragraphs 24-38, wherein the    immune checkpoint protein is selected from the group consisting of:    -   PD-1; PD-L1; PD-L2; TIM-3; CTLA4; TIGIT; DD1-α; A2AR; B7-H3;        B7-H4; BTLA; IDO; TDO; KIR; and LAG3.-   40. The kit or composition of paragraph 39, wherein the inhibitor of    an immune checkpoint protein is selected from the group consisting    of:    -   MGA271; ipilimumab; pembrolizumab; nivolumab; atezolizumab;        galiximab; IMP321; BMS-986016; SMB-663513; PF-05082566; IPH2101;        KW-0761; CDX-1127; MEDI-6769; CP-870,893; tremelimumab;        pidilizumab; MPDL3280A; MEDI4736; MSB0010718C; AUNP12; avelumab;        and durvalumab.-   41. The kit or composition of any of paragraphs 24-40, wherein the    inhibitor of CHI3L1 and the inhibitor of an immune checkpoint    protein are present in the same bivalent antibody reagent.-   42. The kit or composition of paragraph 41, wherein the immune    checkpoint protein is PD-1 or CTLA4.-   43. A therapeutically effective amount of an inhibitor of CHI3L1 and    an inhibitor of an immune checkpoint protein for use in the    treatment of cancer.-   44. The inhibitors of paragraph 43, wherein the inhibitor of CHI3L1    and the inhibitor of an immune checkpoint protein are present in the    same composition.-   45. The inhibitors of paragraph 43, wherein the inhibitor of CHI3L1    and the inhibitor of an immune checkpoint protein are present in    separate compositions.-   46. The inhibitors of any of paragraphs 43-45, wherein the inhibitor    of CHI3L1 is an antibody, antibody reagent, antigen-binding fragment    thereof, or CAR that specifically binds to CHI3L1.-   47. The inhibitors of paragraph 46, wherein an antibody, antibody    reagent, antigen-binding fragment thereof, or CAR comprises at least    one heavy or light chain complementarity determining region (CDR)    selected from the group consisting of:    -   (a) a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 4;    -   (b) a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 5;    -   (c) a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 6;    -   (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 1;    -   (c) a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 2; and    -   (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 3; or

a conservative substitution variant of one or more of (a)-(f).

-   48. The inhibitors of any of paragraphs 46-48, wherein the antibody,    antibody reagent, antigen-binding portion thereof, or CAR of    comprises heavy chain CDRs having the amino acid sequences of SEQ ID    NOs: 1-3 or a conservative substitution variant of such amino acid    sequence.-   49. The inhibitors of any of paragraphs 46-48, wherein the antibody,    antibody reagent, antigen-binding portion thereof or CAR comprises    light chain CDRs having the amino acid sequences of SEQ ID NOs: 4-6    or a conservative substitution variant of such amino acid sequence.-   50. The inhibitors of any of paragraphs 46-49, wherein the antibody,    antibody reagent, antigen-binding portion thereof or CAR comprises    light chain CDRs having the amino acid sequences of SEQ ID NOs: 4-6    and heavy chain CDRs having the amino acid sequences of SEQ ID NOs:    1-3 or a conservative substitution variant of such amino acid    sequence.-   51. The inhibitors of any of paragraphs 43-45, wherein the inhibitor    of CHI3L1 is antibody, antibody reagent, antigen-binding portion    thereof or CAR that competes for binding to CHI3L1 with an antibody    comprising light chain CDRs having the amino acid sequences of SEQ    ID NOs: 4-6 and heavy chain CDRs having the amino acid sequences of    SEQ ID NOs: 1-3.-   52. The inhibitors of any of paragraphs 43-51, wherein the inhibitor    of CHI3L1 binds a CHI3L1 polypeptide at an epitope selected from SEQ    ID NOs: 13-24.-   53. The inhibitors of any of paragraphs 43-52, wherein the inhibitor    of CHI3L1 is an inhibitor of a CHI3L1 receptor.-   54. The inhibitors of paragraph 53, wherein the CHI3L1 receptor is    IL13Rα2, TMEM219, or CRTH2.-   55. The inhibitors of any of paragraphs 43-54, wherein the inhibitor    is an inhibitory antibody or nucleic acid molecule.-   56. The inhibitors of any of paragraphs 43-55, wherein the cancer is    a primary cancer or a metastatic cancer.-   57. The inhibitors of any of paragraphs 43-56, wherein the cancer is    malignant cancer.-   58. The inhibitors of any of paragraphs 43-57, wherein the cancer is    selected from the group consisting of:    -   prostate cancer, colon cancer, rectal cancer, ovarian cancer,        kidney cancer, breast cancer, glioblastoma, melanoma, malignant        melanoma, and lung cancer.-   59. The inhibitors of any of paragraphs 43-58, wherein the inhibitor    of an immune checkpoint protein is an antibody, antibody reagent,    antigen-binding fragment thereof, or CAR that specifically binds to    at least one immune checkpoint protein.-   60. The inhibitors of any of paragraphs 43-59, wherein the inhibitor    of an immune checkpoint protein is a natural ligand thereof-   61. The inhibitors of paragraph 60, wherein the natural ligand    comprises PD-L1 or B7.-   62. The inhibitors of any of paragraphs 43-61, wherein the immune    checkpoint protein is selected from the group consisting of:    -   PD-1; PD-L1; PD-L2; TIM-3; CTLA4; TIGIT; DD1-α; A2AR; B7-H3;        B7-H4; BTLA; IDO; TDO; KIR; and LAG3.-   63. The inhibitors of paragraph 62, wherein the inhibitor of an    immune checkpoint protein is selected from the group consisting of:    -   MGA271; ipilimumab; pembrolizumab; nivolumab; atezolizumab;        galiximab; IMP321; BMS-986016; SMB-663513; PF-05082566; IPH2101;        KW-0761; CDX-1127; MEDI-6769; CP-870,893; tremelimumab;        pidilizumab; MPDL3280A; MEDI4736; MSB0010718C; AUNP12; avelumab;        and durvalumab.-   64. The inhibitors of any of paragraphs 43-63, wherein the inhibitor    of CHI3L1 and the inhibitor of an immune checkpoint protein are    present in the same bivalent antibody reagent.-   65. The inhibitors of paragraph 64, wherein the immune checkpoint    protein is PD-1 or CTLA4.-   66. The inhibitors of any of paragraphs 43-64, wherein the    inhibitors are administered to a subject determined to have an    elevated level of CHI3L1.-   67. The inhibitors of 66, wherein the CHI3L1 is circulating CHI3L1.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   1. A method of treating cancer in a subject in need thereof, the    method comprising administering:    -   a) an inhibitor of an immune checkpoint protein; and.    -   b) at least one agent selected from:        -   i) an inhibitor of Sema7A;        -   ii) an inhibitor of β1-integrin;        -   iii) an agonist of PlexinC1; and        -   iv) an inhibitor of CHI3L1.-   2. The method of paragraph 1, wherein the inhibitor of CHI3L1 is an    antibody, antibody reagent, antigen-binding fragment thereof, or CAR    that specifically binds to CHI3L1.-   3. The method of paragraph 2, wherein an antibody, antibody reagent,    antigen-binding fragment thereof, or CAR comprises at least one    heavy or light chain complementarity determining region (CDR)    selected from the group consisting of:    -   (a) a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 4;    -   (b) a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 5;    -   (c) a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 6;    -   (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 1;    -   (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 2; and    -   (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 3; or

a conservative substitution variant of one or more of (a)-(f).

-   4. The method of paragraph 3, wherein the antibody, antibody    reagent, antigen-binding portion thereof, or CAR of comprises heavy    chain CDRs having the amino acid sequences of SEQ ID NOs: 1-3 or a    conservative substitution variant of such amino acid sequence.-   5. The method of any of paragraphs 3-4, wherein the antibody,    antibody reagent, antigen-binding portion thereof or CAR comprises    light chain CDRs having the amino acid sequences of SEQ ID NOs: 4-6    or a conservative substitution variant of such amino acid sequence.-   6. The method of any of paragraphs 3-5, wherein the antibody,    antibody reagent, antigen-binding portion thereof or CAR comprises    light chain CDRs having the amino acid sequences of SEQ ID NOs: 4-6    and heavy chain CDRs having the amino acid sequences of SEQ ID NOs:    1-3 or a conservative substitution variant of such amino acid    sequence.-   7. The method of paragraph 2, wherein the inhibitor of CHI3L1 is    antibody, antibody reagent, antigen-binding portion thereof or CAR    that competes for binding to CHI3L1 with an antibody comprising    light chain CDRs having the amino acid sequences of SEQ ID NOs: 4-6    and heavy chain CDRs having the amino acid sequences of SEQ ID NOs:    1-3.-   8. The method of any of paragraphs 1-7, wherein the inhibitor of    CHI3L1 binds a CHI3L1 polypeptide at an epitope selected from SEQ ID    NOs: 13-24.-   9. The method of paragraph 1, wherein the inhibitor of CHI3L1 is an    inhibitor of a CHI3L1 receptor.-   10. The method of paragraph 9, wherein the CHI3L1 receptor is    IL13Rα2, TMEM219, or CRTH2.-   11. The method of any of paragraphs 9-10, wherein the inhibitor of    Chi3L1 or the immune checkpoint protein is an inhibitory antibody or    nucleic acid molecule.-   12. The method of any of paragraphs 1-11, wherein the inhibitor of    Sema7A or β1-integrin is an inhibitory antibody reagent or nucleic    acid molecule.-   13. The method of any of paragraph 1-12, comprising administering at    least the inhibitor of Sema7A and the inhibitor of β1-integrin.-   14. The method of paragraph 13, wherein a bivalent antibody reagent    comprises the inhibitor of Sema7A and the inhibitor of β1-integrin.-   15. The method of any of paragraphs 1-14, wherein the agonist of    PlexinC1 is a nucleic acid encoding Growth Arrest Specific 5 (GASS).-   16. The method of any of paragraph 1-15, wherein the cancer is a    primary cancer or a metastatic cancer.-   17. The method of any of paragraphs 1-16, wherein the cancer is    malignant cancer.-   18. The method of any of paragraphs 1-17, wherein the cancer is    selected from the group consisting of:    -   prostate cancer, colon cancer, rectal cancer, ovarian cancer,        kidney cancer, breast cancer, glioblastoma, melanoma, malignant        melanoma, and lung cancer.-   19. The method of any of paragraphs 1-18, wherein the inhibitor of    an immune checkpoint protein is an antibody, antibody reagent,    antigen-binding fragment thereof, or CAR that specifically binds to    at least one immune checkpoint protein.-   20. The method of any of paragraphs 1-19, wherein the inhibitor of    an immune checkpoint protein is a natural ligand thereof.-   21. The method of paragraph 20, wherein the natural ligand comprises    PD-L1 or B7.-   22. The method of any of paragraphs 1-21, wherein the immune    checkpoint protein is selected from the group consisting of:    -   PD-1; PD-L1; PD-L2; TIM-3; CTLA4; TIGIT; DD1-α; A2AR; B7-H3;        B7-H4; BTLA; IDO; TDO; KIR; and LAG3.-   23. The method of paragraph 22, wherein the inhibitor of an immune    checkpoint protein is selected from the group consisting of:    -   MGA271; ipilimumab; pembrolizumab; nivolumab; atezolizumab;        galiximab; IMP321; BMS-986016; SMB-663513; PF-05082566; IPH2101;        KW-0761; CDX-1127; MEDI-6769; CP-870,893; tremelimumab;        pidilizumab; MPDL3280A; MEDI4736; MSB0010718C; AUNP12; avelumab;        and durvalumab.-   24. The method of any of paragraphs 1-23, wherein a) the inhibitor    of CHI3L1, the inhibitor of Sema7A, the inhibitor of β1-integrin,    and/or the agonist of PlexinC1 and b) the inhibitor of an immune    checkpoint protein are present in the same bivalent antibody    reagent.-   25. The method of paragraph 24, wherein the immune checkpoint    protein is PD-1 or CTLA4.-   26. The method of any of paragraphs 1-25, wherein the subject is a    subject determined to have an elevated level of CHI3L1.-   27. The method of paragraph 26, wherein the CHI3L1 is circulating    CHI3L1.-   28. A bi-specific antibody reagent comprising    -   a) an inhibitor of CHI3L1, an inhibitor of Sema7A, an inhibitor        of β1-integrin, and/or an agonist of PlexinC1; and    -   b) an inhibitor of an immune checkpoint protein.-   29. A bi-specific antibody reagent comprising    -   an inhibitor of Sema7A; and    -   an inhibitor of β1-integrin.-   30. A pharmaceutical composition comprising    -   a) an inhibitor of an immune checkpoint protein; and.    -   b) at least one agent selected from:        -   i) an inhibitor of Sema7A;        -   ii) an inhibitor of β1-integrin;        -   iii) an agonist of PlexinC1; and        -   iv) an inhibitor of CHI3L1.-   31. A kit comprising    -   a) a pharmaceutical composition comprising an inhibitor of an        immune checkpoint protein; and.    -   b) at least one pharmaceutical composition comprising at least        one agent selected from:        -   i) an inhibitor of Sema7A;        -   ii) an inhibitor of β1-integrin;        -   iii) an agonist of PlexinC1; and        -   iv) an inhibitor of CHI3L1.-   32. The kit or composition of any of paragraphs 30-32, wherein the    inhibitor of CHI3L1 is an antibody, antibody reagent,    antigen-binding fragment thereof, or CAR that specifically binds to    CHI3L1.-   33. The kit or composition of paragraph 32, wherein an antibody,    antibody reagent, antigen-binding fragment thereof, or CAR comprises    at least one heavy or light chain complementarity determining region    (CDR) selected from the group consisting of:    -   (a) a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 4;    -   (b) a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 5;    -   (c) a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 6;    -   (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 1;    -   (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 2; and    -   (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 3; or

a conservative substitution variant of one or more of (a)-(f).

-   34. The kit or composition of any of paragraphs 32-33, wherein the    antibody, antibody reagent, antigen-binding portion thereof, or CAR    of comprises heavy chain CDRs having the amino acid sequences of SEQ    ID NOs: 1-3 or a conservative substitution variant of such amino    acid sequence.-   35. The kit or composition of any of paragraphs 32-34, wherein the    antibody, antibody reagent, antigen-binding portion thereof or CAR    comprises light chain CDRs having the amino acid sequences of SEQ ID    NOs: 4-6 or a conservative substitution variant of such amino acid    sequence.-   36. The kit or composition of any of paragraphs 32-35, wherein the    antibody, antibody reagent, antigen-binding portion thereof or CAR    comprises light chain CDRs having the amino acid sequences of SEQ ID    NOs: 4-6 and heavy chain CDRs having the amino acid sequences of SEQ    ID NOs: 1-3 or a conservative substitution variant of such amino    acid sequence.-   37. The kit or composition of any of paragraphs 32-36, wherein the    inhibitor of CHI3L1 is antibody, antibody reagent, antigen-binding    portion thereof or CAR that competes for binding to CHI3L1 with an    antibody comprising light chain CDRs having the amino acid sequences    of SEQ ID NOs: 4-6 and heavy chain CDRs having the amino acid    sequences of SEQ ID NOs: 1-3.-   38. The kit or composition of any of paragraphs 32-37, wherein the    inhibitor of CHI3L1 binds a CHI3L1 polypeptide at an epitope    selected from SEQ ID NOs: 13-24.-   39. The kit or composition of any of paragraphs 32-38, wherein the    inhibitor of CHI3L1 is an inhibitor of a CHI3L1 receptor.-   40. The kit or composition of paragraph 39, wherein the CHI3L1    receptor is IL13Rα2, TMEM219, or

CRTH2.

-   41. The kit or composition of any of paragraphs 30-31, wherein the    inhibitor of Chi3L1 or the immune checkpoint inhibitor is an    inhibitory antibody or nucleic acid molecule.-   42. The kit or composition of any of paragraphs 30-41, wherein the    inhibitor of Sema7A or β1-integrin is an inhibitory antibody reagent    or nucleic acid molecule.-   43. The kit or composition of any of paragraph 30-42, comprising    administering at least the inhibitor of Sema7A and the inhibitor of    β1-integrin.-   44. The kit or composition of paragraph 43, wherein a bivalent    antibody reagent comprises the inhibitor of Sema7A and the inhibitor    of β1-integrin.-   45. The kit or composition of any of paragraphs 30-44, wherein the    agonist of PlexinC1 is a nucleic acid encoding Growth Arrest    Specific 5 (GASS).-   46. The kit or composition of any of paragraphs 30-45, wherein the    inhibitor of an immune checkpoint protein is an antibody, antibody    reagent, antigen-binding fragment thereof, or CAR that specifically    binds to at least one immune checkpoint protein.-   47. The kit or composition of any of paragraphs 30-45, wherein the    inhibitor of an immune checkpoint protein is a natural ligand    thereof.-   48. The kit or composition of paragraph 47, wherein the natural    ligand comprises PD-L1 or B7.-   49. The kit or composition of any of paragraphs 30-48 wherein the    immune checkpoint protein is selected from the group consisting of:    -   PD-1; PD-L1; PD-L2; TIM-3; CTLA4; TIGIT; DD1-α; A2AR; B7-H3;        B7-H4; BTLA; IDO; TDO; KIR; and LAG3.-   50. The kit or composition of paragraph 49, wherein the inhibitor of    an immune checkpoint protein is, or comprises the CDRs of an agent,    selected from the group consisting of:    -   MGA271; ipilimumab; pembrolizumab; nivolumab; atezolizumab;        galiximab; IMP321; BMS-986016; SMB-663513; PF-05082566; IPH2101;        KW-0761; CDX-1127; MEDI-6769; CP-870,893; tremelimumab;        pidilizumab; MPDL3280A; MEDI4736; MSB0010718C; AUNP12; avelumab;        and durvalumab.-   51. The kit or composition of any of paragraphs 30-50 wherein the    inhibitor of CHI3L1 and the inhibitor of an immune checkpoint    protein are present in the same bivalent antibody reagent.-   52. The kit or composition of paragraph 51, wherein the immune    checkpoint protein is PD-1 or CTLA4.-   53. A therapeutically effective amount of    -   a) an inhibitor of an immune checkpoint protein; and.    -   b) at least one agent selected from:        -   i) an inhibitor of Sema7A;        -   ii) an inhibitor of β1-integrin;        -   iii) an agonist of PlexinC1; and        -   iv) an inhibitor of CHI3L1.

for use in the treatment of cancer.

-   54. The agents of paragraph 53, wherein the at least one agent of b)    and the inhibitor of an immune checkpoint protein are present in the    same composition.-   55. The agents of paragraph 53, wherein the at least one agent of b)    and the inhibitor of an immune checkpoint protein are present in    separate compositions.-   56. The agents of any of paragraphs 53-55, wherein the inhibitor of    CHI3L1 is an antibody, antibody reagent, antigen-binding fragment    thereof, or CAR that specifically binds to CHI3L1.-   57. The agents of paragraph 56, wherein an antibody, antibody    reagent, antigen-binding fragment thereof, or CAR comprises at least    one heavy or light chain complementarity determining region (CDR)    selected from the group consisting of:    -   (a) a light chain CDR1 having the amino acid sequence of SEQ ID        NO: 4;    -   (b) a light chain CDR2 having the amino acid sequence of SEQ ID        NO: 5;    -   (c) a light chain CDR3 having the amino acid sequence of SEQ ID        NO: 6;    -   (d) a heavy chain CDR1 having the amino acid sequence of SEQ ID        NO: 1;    -   (e) a heavy chain CDR2 having the amino acid sequence of SEQ ID        NO: 2; and    -   (f) a heavy chain CDR3 having the amino acid sequence of SEQ ID        NO: 3; or

a conservative substitution variant of one or more of (a)-(f).

-   58. The agents of any of paragraphs 53-57, wherein the antibody,    antibody reagent, antigen-binding portion thereof, or CAR of    comprises heavy chain CDRs having the amino acid sequences of SEQ ID    NOs: 1-3 or a conservative substitution variant of such amino acid    sequence.-   59. The agents of any of paragraphs 53-58, wherein the antibody,    antibody reagent, antigen-binding portion thereof or CAR comprises    light chain CDRs having the amino acid sequences of SEQ ID NOs: 4-6    or a conservative substitution variant of such amino acid sequence.-   60. The agents of any of paragraphs 53-59, wherein the antibody,    antibody reagent, antigen-binding portion thereof or CAR comprises    light chain CDRs having the amino acid sequences of SEQ ID NOs: 4-6    and heavy chain CDRs having the amino acid sequences of SEQ ID NOs:    1-3 or a conservative substitution variant of such amino acid    sequence.-   61. The agents of any of paragraphs 53-60, wherein the inhibitor of    CHI3L1 is antibody, antibody reagent, antigen-binding portion    thereof or CAR that competes for binding to CHI3L1 with an antibody    comprising light chain CDRs having the amino acid sequences of SEQ    ID NOs: 4-6 and heavy chain CDRs having the amino acid sequences of    SEQ ID NOs: 1-3.-   62. The agents of any of paragraphs 53-61, wherein the inhibitor of    CHI3L1 binds a CHI3L1 polypeptide at an epitope selected from SEQ ID    NOs: 13-24.-   63. The agents of any of paragraphs 53-62, wherein the inhibitor of    CHI3L1 is an inhibitor of a CHI3L1 receptor.-   64. The agents of paragraph 63, wherein the CHI3L1 receptor is    IL13Rα2, TMEM219, or CRTH2.-   65. The agents of any of paragraphs 53-64, wherein the inhibitor of    Chi3L1 or the immune checkpoint protein is an inhibitory antibody or    nucleic acid molecule.-   66. The agents of any of paragraphs 53-65, wherein the inhibitor of    Sema7A or β1-integrin is an inhibitory antibody reagent or nucleic    acid molecule.-   67. The agents of any of paragraphs 53-66, comprising both the    inhibitor of Sema7A and the inhibitor of β1-integrin.-   68. The agents of paragraph 67, wherein a bivalent antibody reagent    comprises the inhibitor of Sema7A and the inhibitor of β1-integrin.-   69. The agents of any of paragraphs 53-68, wherein the agonist of    PlexinC1 is a nucleic acid encoding Growth Arrest Specific 5 (GASS).-   70. The agents of any of paragraphs 53-69, wherein the cancer is a    primary cancer or a metastatic cancer.-   71. The agents of any of paragraphs 53-70, wherein the cancer is    malignant cancer.-   72. The agents of any of paragraphs 53-71, wherein the cancer is    selected from the group consisting of:    -   prostate cancer, colon cancer, rectal cancer, ovarian cancer,        kidney cancer, breast cancer, glioblastoma, melanoma, malignant        melanoma, and lung cancer.-   73. The agents of any of paragraphs 53-72, wherein the inhibitor of    an immune checkpoint protein is an antibody, antibody reagent,    antigen-binding fragment thereof, or CAR that specifically binds to    at least one immune checkpoint protein.-   74. The agents of any of paragraphs 53-73, wherein the inhibitor of    an immune checkpoint protein is a natural ligand thereof-   75. The agents of paragraph 74, wherein the natural ligand comprises    PD-L1 or B7.-   76. The agents of any of paragraphs 53-75, wherein the immune    checkpoint protein is selected from the group consisting of:    -   PD-1; PD-L1; PD-L2; TIM-3; CTLA4; TIGIT; DD1-α; A2AR; B7-H3;        B7-H4; BTLA; IDO; TDO; KIR; and LAG3.-   77. The agents of paragraph 76, wherein the inhibitor of an immune    checkpoint protein is selected from the group consisting of:    -   MGA271; ipilimumab; pembrolizumab; nivolumab; atezolizumab;        galiximab; IMP321; BMS-986016; SMB-663513; PF-05082566; IPH2101;        KW-0761; CDX-1127; MEDI-6769; CP-870,893; tremelimumab;        pidilizumab; MPDL3280A; MEDI4736; MSB0010718C; AUNP12; avelumab;        and durvalumab.-   78. The agents of any of paragraphs 53-77, wherein the inhibitor of    CHI3L1 and the inhibitor of an immune checkpoint protein are present    in the same bivalent antibody reagent.-   79. The agents of paragraph 78, wherein the immune checkpoint    protein is PD-1 or CTLA4.-   80. The agents of any of paragraphs 53-79, wherein the agents are    administered to a subject determined to have an elevated level of    CHI3L1.-   81. The agents of 80, wherein the CHI3L1 is circulating CHI3L1.

EXAMPLES Example 1: Chitinase 3-Like-1 (Chi3l1) Neutralizing Antibodiesas Therapeutics in Asthma and Lung Cancer

Members of the 18 glycosyl hydrolase (GH) gene family are dysregulatedin and play an important role in the pathogenesis of a variety ofdiseases. This is particularly striking for the chitinase-like proteincalled chitinase 3-like-1 (Chi3l1; also called Chil1 in mice and YKL-40in man) in asthma and lung cancer.

It has been demonstrated that the levels of circulating Chi3l1 areincreased in human asthma where they correlate with disease severity(1-4). Single nucleotide polymorphisms of Chi3l1 that correlate withincreased levels of circulating Chi3l1, asthma prevalence and poor lungfunction have been identified (2, 3, 5). In accord with the items notedabove, it was also demonstrated that null mutations of Chi3l1 markedlydecreased Th2 inflammation and eosinophil accumulation in aeroallergenmurine asthma models (6).

The levels of circulating YKL-40 are increased in many malignanciesincluding cancers of the prostate, colon, rectum, ovary, kidney, breast,glioblastomas and malignant melanoma (7-19). In these diseases, thelevels of YKL-40 frequently correlate directly with disease progressionand inversely with disease-free interval and survival (7-19). This isparticularly striking in lung cancer where the serum and tissue levelsof YKL-40 are impressively increased and correlate with adverse outcomes(20-23). To address the roles of Chi3l1 in these responses, the roles ofChi3l1 in primary and metastatic lung cancer were evaluated. Thesestudies demonstrate that (1) YKL-40 is expressed in an exaggeratedmanner in human lung cancer where it correlates inversely with survival;(2) in murine models, Chi3l1 is sequentially induced in normal peritumorand tumor tissues during the early and later stages, respectively, oflung cancer development; (3) Chi3l1 induction via a semaphorin7a-dependent mechanism plays a critical role in the generation of ametastasis permissive pulmonary microenvironment; (4) in metastaticmodels, Chi3l1 production and metastatic spread can be inhibited viaRIG-like helicase (RLH) innate immunity (24, 25). These studiesdemonstrate that Chi3l1 is induced in selective tissue compartmentsduring lung cancer initiation and progression and define the essentialrole that it plays in disease progression.

Generation and Characterization of Antibody-Based Anti-Chi3l1 BasedTherapeutics.

Because Chi3l1 is induced in patients with asthma and lung cancer andplays a critical role in the pathogenesis of murine models of bothdiseases studies were undertaken to develop and assess the efficacy ofChi3l1 neutralizing antibodies. Epitopes of Chi3l1 were selected (seeTables 1 and 2 for epitope design and selection) and monoclonalantibodies were generated.

The antibodies were then assessed for their (a) sensitivity, andspecificity of detection of Chi3l1 in human and murine systems usingdenatured and non-denatured Chi3l1; (b) binding affinity; (c) ability toneutralize the effects of rChi3l1 in cell based assays; and (d) abilityto neutralize Chi3l1 in vivo. Among the antibodies that were generatedone, called FRG, had the most exciting characteristics. It is an IgG2bKappa antibody that powerfully detects human and murine Chi3l1 underdenaturing and non-denaturing conditions with high specificity (FIGS.1A-1D). It also blocks rChi3l1 induced MAPK and AKT signaling in vitro(FIGS. 2A-2B).

To determine if FRG blocked asthma-like inflammation, the ovalbuminsensitization and challenge murine model was utilized. Wild type miceand mice in which murine Chi3l1 (Chil1) was overexpressed in the airwayusing the CC10 (also called CCSP) promoter were employed. The effects onairway eosinophilic inflammation were assessed in mice that received anIgG2b control antibody or FRG at a dose of 200 μg/mouse every other day.As can be seen in FIG. 3, the overexpression of Chil1 (Chi3l1) under theinfluence of the CC10 promoter augmented ovalbumin sensitization andchallenge-induced eosinophil recovery as seen in FIG. 3. As can also beseen in FIG. 3, FRG was a potent inhibitor of aeroallergen-inducedeosinophilic inflammation and BAL eosinophil accumulation.

To determine if FRG blocked metastatic cancer B16-F10 malignantmelanocytes were used as described previously (24, 25). Varying numbersof these cells were administered by tail vein to wild type mice andpulmonary metastasis were quantitated. Melanoma metastasis were assessedin mice that received an IgG2b control antibody or FRG at a dose of200m/mouse every other day. As can be seen in FIG. 4, FRG was a potentinhibitor of melanoma metastasis. Importantly, these effects were notrestricted to melanoma because similar results were seen with breastcancer cells.

Studies were next undertaken to determine if FRG ameliorated thegeneration of primary lung cancer. In these studies, primary lungcancers were induced in mice with KRAS^(G12D) mutations and p53mutations (26, 27) and tumor generation was characterized. In this modela discrete mass of tumor cells and a macrophage-rich inflammatoryresponse can be seen 6 weeks after Adeno-Cre recombinase challenge thatgradually increases over time (FIG. 5). The generation of primary lungcancers were assessed in mice that received an IgG2b control antibody orFRG at a dose of 200m/mouse every other day. As can be seen in FIGS.5A-5B, FRG was a potent inhibitor of the generation of primary lungcancers in this model.

Because of the impressive results of FRG in these in vivo models itsvariable regions were sequenced. The sequences are noted in Table 3 andthe heavy and light chain CDRs are depicted in FIGS. 7 and 8. Thelocation of selected epitopes including that recognized by FRG in humanChi3l1 are depicted in FIG. 6.

B16F10 melanoma metastasis in mice was treated with the FRG antibodydescribed herein in combination with Anti-PD-1 (FIG. 9). In theseexperiments malignant melanocytes were administered by tail vein to wildtype (WT) mice that were treated every other day with the noted dosagesof FRG and or anti-PD-1, alone and in combination, via anintraperitoneal route. Fourteen days later the lungs were removed andthe levels of metastasis (black dots on pleural surface) were assessed.The beneficial interactions of FRG and anti-PD-1 in combination can beseen in FIG. 9 and are quantified in FIG. 10.

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TABLE 1 Parameters considered in epitope selection algorithms. SecondaryStructure Amino Acid property Loop/helix/sheet Antigenic enhancementamino acid Special region Flexibility N-terminal, C-terminal EvolutionSignal peptide Positive selection Trans-membran DiscriminationDisordered region Customer requests Solvent accessibility Proteinspecificity Blast Region specificity Query species and Mouse Others

TABLE 2  List of selected epitopes including FRG (ID Number 0) ID numberStart End Peptide SEQ ID NO 0 223 234 FRGQEDASPDRF 13 1 304 315RGATVHRILGQQ 14 2 268 279 ASSETGVGAPIS 15 3 162 173 IKEAQPGKKQLL 16 4 6273 SNDHIDTWEWND 17 5 141 152 YPGRRDKQHFTT 18 6 245 256 LRLGAPASKLVM 19 7281 292 PGIPGRFTKEAG 20 8 102 113 GSQRFSKIASNT 21 9 181 192 GKVTIDSSYDIA22 10 78 89 GMLNTLKNRNPN 23 11 111 122 SNTQSRRTFIKS 24

Location of selected epitopes including FRG in human Chi3I1 (italics)

(SEQ ID NO: 25) MGVKASQTGFVVLVLLQCCSAYKLVCYYTSWSQYREGDGSCFPDALDRFLCTHIIYSFANISNDHIDTWEWNDVTLYGMLNTLKNRNPNLKTLLSVGGWNFGSQRFSKIASNTQSRRTFIKSVPPFLRTFIGFDGLDLAWLYPGRRDKQHFTTLIKEMKAEFIKEAQPGKKQLLLSAALSAGKVTIDSSYDIAKISQHLDFISIMTYDFHGAWRGTTGHHSPL

SNTDYAVGYMLRLGAP ASKLVMGIPTFGRSFTLASSETGVGAPISGPGIPGRFTKEAGTLAYYEICDFLRGATVHRILGQQVPYATKGNQWVGYDDQESVKSKVQYLKDRQLAGAMVWALDLDDFQGSFCGQDLRFPLTNAIKDALAAT

TABLE 3  Sequences of variable complementarity determiningregions (CDRs) of FRG antibody SEQ ID NO: Heavy CDR1 GYTFTNYG 1 chain(DNA) (GGGTATACCITCACAAACTATGGA) 7 (IgG2b) CDR2 I N T Y T G E P 2 (DNA)(ATAAATACCTACACTGGAGAGCCA) 8 CDR3 ARLGYGKFYVMDY 3 (DNA)(GCAAGATTGGGATATGGTAAATTCTAG 9 TTATGGACTAC) Light CDR1 QSLVHSNGNTY 4chain (DNA) (CAGAGCCTTGTACACAGTAATGGAAAC 10 (IgG K) ACCTAT) CDR2 K V S 5(DNA) (AAAGTTTCC) 11 CDR3 S Q S T H V T W T 6 (DNA)(TCTCAAAGTACACATGTTACGTGGACG) 12

Example 2

When the FRG antibody was administered in combination with a checkpointinhibitor (e.g., anti-PD-1), synergism was observed (FIGS. 9, 10), withthe combination displaying improved efficacy in reducing B16F10metastasis. Accordingly, it is contemplated herein that the combinationof CHI3L1 inhibition and inhibition of a checkpoint protein providessynergistic efficacy in the treatment of cancer.

It is specifically contemplated herein that CHI3L1 functions as aregulator of immune checkpoint proteins and costimulatory molecules on Tcells. Accordingly, targeting both CHI3L1 and a checkpoint proteinprovides a synergistic effect.

1-29. (canceled)
 30. A method of treating cancer in a subject in needthereof, the method comprising administering bispecific antibody thatdetects and neutralizes chitinase 3-like-1 (CHI3L1) and programed deathreceptor 1 (PD 1) comprising: (a) an antigen-binding portion of ananti-human PD1 antibody; and (b) an antigen-binding portion of ananti-human CHI3L1 antibody comprising light chain complementaritydetermining regions (CDRs) having the amino acid sequences of SEQ IDNOs: 4-6 and heavy chain CDRs having the amino acid sequences of SEQ IDNOs: 1-3, wherein the cancer expresses PD-L1 and CHI3L1.
 31. The methodof claim 30, wherein the bispecific antibody comprises an anti-humanCHI3L1 single chain variable fragment (ScFv CHI3L1) attached to abackbone of an anti-human PD-1 antibody.
 32. The method of claim 30,wherein the bispecific antibody comprises an anti-human PD-1 singlechain variable fragment (ScFv PD1) attached to a backbone of ananti-human CHI3L1 antibody.
 33. The method of claim 32, wherein the ScFvPD1 is attached to the heavy chain of the CHI3L1 antibody backbone. 34.The method of claim 32, wherein the ScFv PD1 is attached to the lightchain of the CHI3L1 antibody backbone.
 35. The method of claim 32,wherein the anti-human CHI3L1 antibody comprises a heavy chain sequencehaving the amino acid sequence of SEQ ID NO: 36 or a light chainsequence having the amino acid sequence of SEQ ID NO:
 38. 36. The methodof claim 32, wherein the anti-human CHI3L1 antibody comprises a heavychain sequence having the amino acid sequence of SEQ ID NO: 36 and alight chain sequence having the amino acid sequence of SEQ ID NO: 38.37. The method of claim 32, wherein the anti-human CHI3L1 antibodycomprises a conservative substitution relative to the heavy chainsequence having the amino acid sequence of SEQ ID NO: 36 or the lightchain sequence having the amino acid sequence of SED ID NO: 38, whereinthe conservative substitution is in a sequence not comprised by a CDR.38. The method of claim 30, wherein the antigen-binding portion of theanti-human CHI3L1 antibody binds a CHI3L1 polypeptide at an epitopehaving the amino acid sequences of SEQ ID NO:
 13. 39. The method ofclaim 30, wherein the cancer is a primary cancer or a metastatic cancer.40. The method of claim 30, wherein the cancer is malignant cancer. 41.The method of claim 30, wherein the cancer is selected from the groupconsisting of: prostate cancer, colon cancer, rectal cancer, ovariancancer, kidney cancer, breast cancer, glioblastoma, melanoma, malignantmelanoma, and lung cancer.
 42. The method of claim 30, wherein thesubject is a subject determined to have an elevated level of CHI3L1compared to a reference level.
 43. The method of claim 42, wherein theCHI3L1 is circulating CHI3L1.
 44. The method of claim 30, wherein thesubject is a subject determined to have a level of CHI3L1 that isincreased compared to a prior assessment of the level in that subject.45. The method of claim 44, wherein the CHI3L1 is circulating CHI3L1.